CZ298353B6 - Non-viral vector - Google Patents

Abstract

In the present invention, there is disclosed a nucleic acid delivery complex comprising a condensed polypeptide/nucleic acid complex and a cationic lipid wherein the complex comprises (a) a nucleic acid sequence of interest; and (b) one or more viral nucleic acid packaging polypeptides, or derivatives thereof, said polypeptides or derivatives thereof being (i) capable of binding to the nucleic acid sequence of interest; and (ii) capable of condensing the nucleic acid sequence of interest; and wherein the nucleic acid sequence of interest is heterologous with respect to the polypeptide. There is also disclosed a method of introducing a nucleic acid sequence of interest into a cell through the mediation of a delivery vector.

Description

The present invention relates to cationic lipid / protein / nucleic acid complexes containing viral packaging proteins, and their use in the efficient delivery of nucleic acids to cells such as neuronal cells.

BACKGROUND OF THE INVENTION

Promising advances in non-viral gene transfer have been achieved due to the production of synthetic liposomes formulated with cationic lipids that are capable of transfecting cells. However, few of these complexes have been tested for the ability to efficiently transfer DNA into CNS cells to obtain transgene expression. The ability to efficiently and safely transfect neuronal cells could provide a powerful tool to elucidate neuronal function and could lead to new ways of treating nerve disorders.

However, the CNS gene therapy could not be fully utilized due to the lack of an effective means for transduction of post-mitotic neurons. Most studies have been used to deliver viral vector genes. However, a disadvantage of many viral vectors is the immunity and cytotoxicity problems and the difficulty of manipulation by persons who are not experts in virology (Wood MJA et al., Gene Ther. 1994; 1 Neuroscience 1995; 66: 1015 - 1024; Naldini L. et al., In vivo Gene Delivery and Stable Transduction of Nondiving Cell and Lentiviral Vector [See: 283-291; Byrnes AP et al., Adenovirus gene-transfer causes inflammation in the brain. Science 1996; 272: 263-267). Non-viral vectors are now emerging as an alternative method for cell transduction. The most promising advances in non-viral gene transfer have been achieved in the production of synthetic liposomes formulated with cationic lipids (cytofectins) capable of transfecting cells. These cationic liposomes are relatively easy to use, have wide utility and lack cytotoxicity (Miller, A. D., Cationic liposomes for gene delivery. Angewandte Chemie International Edition 1998; 37: 1769-1785).

New formulations of cationic liposomes are currently under development (Lee RJ, Huang L., Lipidic vector systems for gene transfer. Critical Reviews in Therapeutic Drug Carrier Systems 1997; 14: 173-206). However, only a small number of these complexes have been tested for the ability to efficiently transduce cells within the CNS (Gao X., Huang L. ₎ Cationic liposome-mediated genetransfer. Gene Ther. 1995; 2: 710-722; Felgner PL et al., Lipofection- and Highly Efficient, Lipid-Mediated DNA-Transfection Procedure: Proceedings Og, National Academy of Sciences of the United States Of America 1987; 84: 7413-7417; Farhood H., Serbina N., Huang L., The Role of Dioleoyl Phosphatidylethanolamine in Cationic Liposome Mediated Gene transfer, Biochem. Biphys. Acta 1995; 1235: 289-295; Caplen NJ et al., In-vitro liposome-mediated DNA transfection of epithelial-cell lineage using the cationic liposome DC-Chol / DOPE. Gene Ther. 1995; 2: 603-613). Cationic liposomes act through electrostatic interactions with negatively charged DNA followed by cell membranes, passing through the cell membrane through a slow endocytosis process (Gao X., Huang L., Cationic liposome-mediated gene-transfer. Gene Ther. 1995; 2: 710 - 722; Labat-Moleur F. et al., An electron microscopy study into the mechanism of gene transfer with lipopolyamines Gene Ther. 1996; 3: 1010 - 1017; Zabner J. et al., Cellular and molecular barriers to gene transfer by cationic lipid (J. Biol. Chem. 1995; 270: 18997-19897). These liposomes are often formulated using the neutral lipid dioleoyl-La-phosphatidylethanolamine (DOPE), which is extremely effective in buffering and disrupting endosomes (Farhood H., Serbina N., Huang L., The role of dioleoyl phosphatidylethanolamine in cationic liposome mediated gene. Biochem. Biophys. Acta 1995; 1235: 289-295; Felgner JH et al., Enhanced Gene Delivery and Mechanism Studies With a Novel Series of Cationic Lipid Formulations (J. Biol. Chem. 1994; 269: 2550-2561). Transfected genetic material is released from the perinuclear space

From the liposome complex, transported to the nucleus and expressed. So far, it has been shown that only liposomes formulated from N- [1- (2,3-dioleyloxy) propyl] - N, N, N-trimethylammonium chloride (DOTMA) and DOPE (Sahenk Z. et al., J

Delivery to Spinal Motor Neurons. Brain Res. 1993: 606: 126-129; Iwamoto Y. et al., I

Liposome-Mediated Bdnf Cdna Transfer in Intact and Injected Rat-Brain. Neuroreport 1996; 7: j

609-612; Roessler B.J., Davidson B.L., Direct plasmid-mediated transfection of adult murine l brain-cells in-vivo using cationic liposomes. Neurosci Fly. 1994; 167: 5-10; Zhou X., Huang

L., DNA transfection mediated by cationic liposomes containing lipopolylysine: characterization and mechanism of action, Biochim. Biophys. Acta 1994; 1189: 195-203). Liposome complexes capable of highly efficient transfection of CNS cells are highly needed for use in gene therapy.

The major limitation of non-viral gene transfer is the formation of large aggregated molecules in the formation of liposome: DNA complexes (Lee R.J., Huang L., Lipidic vector systems for gene transfer. Critical Reviews in Therapeutic Drug Carrier Systems 1997; 14: 173-206). These large aggregates may reduce transfection efficiency, probably due to a reduction in endocytosis of complexes. One approach to limiting this phenomenon is to reduce the size of the DNA molecules through condensation of the DNA prior to complex formation. DNA precondensation provides smaller complexes and improved transfection efficiency (Sorgi FL, Bhattacharya S., Huang L., Protamine Sulfate Lipid-Mediated Gene Transfer Enhancements. Gene Ther, 1996; 4: 961-968; Li S., Huang L., Protamine Sulfate provides enhanced and reproducible intravenous gene transfer by cationic liposome / DNA complex Joumal of Liposome Research 1997: 7: 207-219; Vitiello L. et al., Condensation of Plasmid DNA with Lysine Polys improves liposome-mediated gene transfer into established and primary Gene Ther. 1996; 3: 396-404; Namiki Y., Takahashi T ,, Ohno T., Gene transduction for disseminated intraperitoneal tumor using cationic liposomes containing non-histoone chromatin proteins: cationic liposomal gene therapy of carcinomatosis. Ther. 1998; 4: 240 246; Gao X., Huang L., Potentiation of cationic liposome-mediated gene delivery by polycations. Biochem. 1996; 35: 9286- 9286; Fritz JD et al., Gene transfer into mammalian cells us The histone-condensed plasmid DNA. Hum. Gene Ther. 1996; 7: 1395-1404; Hagstrom J. E. et al., Complexes of non-cationic liposomes and histone HI mediate efficient transfection of DNA without encapsulation. Biochim. Biophys Acta 1996; 1284: 47-55). Various polycations have been identified that are effective in improving liposome-mediated transfection. Of these substances, L-lysine and protamine poles gave the most significant results, allowing for more than thirty-fold increases compared to non-precondensation complexes in a number of non-neuronal cell lines (Sorgi FL, Bhattacharya S., Huang L., Protamine sulfate enhances lipid-mediated Gene Ther. 1997; 4: 961-968; GaoX., Huang L., Potentiation of cationic liposome-mediated gene delivery by polycations, Biochem. 1996: 35: 9286-9286).

Protamine sulphate has particularly good results in increasing liposome transfection. Protamine is a naturally occurring polycation found in the head of the spermatozoa. The role of protamine is to condense the DNA in the semen and facilitate its transfer to the nucleus of the egg. The core-targeting feature of protamine makes it particularly suitable for gene transfer. Unlike synthetic poly-L-lysine, which has a large molecular weight range (18,000 to 19,200 Da), protamine occurs naturally, is smaller, and has a smaller molecular weight range (4000 to 4250 Da). These properties mean that there is less chance of immunogenic responses in the target tissue and it is easier to control condensation. Other naturally occurring condensing DNA proteins have also been used to increase DNA transfer through liposomes. Fritz et al. (Fritz J. D, et al., Gene Transfer to Mammalian Cells Using Histoone-Condensed Plasmid DNA. Hum. Gene Ther. 1996; 7: 1395-1404) achieved an approximately 30-fold increase in lipofection using recombinant human histone H1 protein carrying a core localization signal (nls-H1). It has also been shown that lipofection is enhanced by a non-histone chromosomal high-mobility group 1,2 protein, which is routinely used in the HVJliposome method (Namiki Y., Takahashi T, Ohno T., Gene transduction for disseminated intraperitoneal tumor using non-histone cationic liposomes) chromatin proteins: cationic liposomal gene therapy of carcinomatosis (Gene Ther, 1998; 5: 240-246; Isaka Y. et al., The HVJ liposome method. Exp-Nephrol 1998; 6: 144-147).

-2GB 298353 B6

SUMMARY OF THE INVENTION

The inventors tested proteins associated with viral DNA for their ability to enhance liposome-based gene transfer. In particular, the authors compared the virally encoded synthetic peptide Mul and recombinant Vpl protein of adenovirus and polyomavirus, respectively. The Mul peptide may play a role in adenovirus chromosome condensation, while VP1 is the only polyomavirus structural protein that exhibits DNA binding activity (Gillock E, T. et al., Polyomavirus major capsid VP1 protein is capable of packaging cellular DNA when expressed in the baculovirus system. J. Virol. 1997; 71: 2857 - 2865 issn: 0022-2538x; Chang D., Cai X., Consigli R, A., Characterization of DNA binding properties of polyomavirus capsid proteins Journal of Virology 1993; 67: 6327-6331; Anderson CW, Young, E. E., Flint SJ, Characterization of the adenovirus 2 virion protein, mu Virology 1989; 172: 506-512). Vpl, but not Mul, contains an embedded classical nuclear localization signal (NLS) similar to that found in HMG-1,2 and nls-H1 (Chang D., Cai X., Consigli RA, Characterization of the DNA binding properties of polyomavirus capsid proteins, Journal of Virology 1993; 67: 6327-6331). The present inventors have found that Mul, but not Vp1, significantly improves cationic liposome-mediated gene transfer to nerve and kidney-derived cells. The inventors also found that the increase by Mul was greater in differentiated cells, suggesting the possible applicability of this approach for neuronal cells in vivo.

These findings are the basis for experimental and therapeutic uses of DNA delivery to liposome-mediated CNS cells.

Accordingly, the present invention provides a non-viral nucleic acid delivery vector comprising a fused polypeptide / nucleic acid complex and a cationic lipid, the complex comprising:

(a) a selected nucleic acid sequence of interest (NOI); and (b) one or more viral nucleic acid packaging polypeptides or derivatives thereof, wherein the polypeptides or derivatives thereof are capable of (i) binding to a selected nucleic acid sequence; and (ii) condense the selected nucleic acid sequence; and wherein the selected nucleic acid sequence is heterologous to the polypeptides.

Preferably, the at least one polypeptide is an adenoviral nucleic acid packaging polypeptide or derivative thereof. More preferably, the adenoviral polypeptide is Mul, pV or pVII, or a derivative thereof.

The term "heterologous to polypeptides" means that viral nucleic acid sequences that naturally occur in combination with a viral packaging polypeptide are excluded.

In a preferred embodiment, the vector further comprises a polypeptide comprising a nuclear localization sequence (NLS). More preferably, the polypeptide comprising the nuclear localization sequence (NLS) is an adenoviral pV or derivative thereof.

The present invention also provides a fused polypeptide / nucleic acid complex comprising a cationic lipid, a polypeptide component, and a nucleic acid component, for use in delivering a nucleic acid component to the nucleus of a eukaryotic cell, wherein (i) the polypeptide component is a viral nucleic acid packaging polypeptide or derivative thereof;

(ii) the polypeptide component or derivative thereof is capable of binding to a selected nucleic acid sequence; and (iii) the polypeptide component or derivative thereof is capable of condensing a selected nucleic acid sequence;

And wherein the nucleic acid is heterologous to the polypeptide.

The at least one polypeptide is preferably an adenoviral nucleic acid packaging polypeptide or derivative thereof. More preferably, the adenoviral polypeptide is Mul, pV or pVII, or a derivative thereof

In a preferred embodiment, the complex further comprises a polypeptide comprising a nuclear localization sequence (NLS). More preferably, the polypeptide comprising the nuclear localization (NLS) sequence is an adenoviral pV or derivative thereof.

The present invention also provides a method for producing a non-viral nucleic acid delivery vector comprising a fused polypeptide / nucleic acid complex and a cationic lipid, comprising the steps of *.

(a) contacting the selected nucleic acid sequence with a viral nucleic acid packaging polypeptide or derivative thereof, wherein the polypeptide component or derivative thereof is capable of (i) binding to the selected nucleic acid sequence; and (ii) condense the selected nucleic acid sequence; and wherein the selected nucleic acid sequence is heterologous to the polypeptide; and (b) contacting the nucleic acid / polypeptide complex so formed with a cationic lipid.

The present invention further provides a method of introducing a selected nucleic acid sequence into a eukaryotic cell, the method comprising contacting the cell with a complex of the invention, wherein the complex comprises the selected nucleic acid sequence. Preferably, the cell is a neuronal, cancer or epithelial cell.

In an alternative embodiment, a viral nucleic acid localization / delivery polypeptide may be used in place of or in addition to the viral nucleic acid packaging polypeptide. However, some viral polypeptides combine both of these functions.

DETAILED DESCRIPTION OF THE INVENTION

Although the techniques herein are well known in the art, reference is made in particular to Sambrook et al., Molecular Cloning, A Laboratory Manual (1989) and Ausubel et al., Short Protocols in Molecular Biology (1999) 4th Edition, John Wiley & Sons, ln.

A. Polypeptide Components

Viral packaging nucleic acid polypeptides

The term "viral nucleic acid packaging polypeptides" typically includes polypeptides encoded by viral genomes that naturally occur in viral particles where their function is to fold, particularly condense, and deliver to the nucleic acid core to form a virion genome in the form of a virion. Also included are its homologues and derivatives such as fragments as set forth below.

Examples of viral nucleic acid packaging polypeptides include viral core proteins such as hepatitis B nuclear antigen and adenoviral nuclear proteins, Mul, pV and pVII, and their equivalent proteins in other adenoviruses such as mastadenoviruses (mammalian adenoviruses) and aviadenoviruses (avian adenoviruses). A particularly preferred viral nucleic acid packaging polypeptide for use in the present invention is the Mul polypeptide shown below as SEQ ID NO. 1.

NH ₂ -Met-Arg-Arg-Ala-His-His-Arg-Arg-Arg-Arg-Arg-Ala-Ser-His-Arg-Arg-Met-ArgGly-Gly-OH (SEQ ID No. 1).

-4GB 298353 B6

The viral nucleic acid packaging polypeptide for use in the present invention is capable of binding to nucleic acids, typically in a non-specific manner, and preferably causes nucleic acid condensation. In general, it is preferred that the condensed selected nucleic acid sequence have a size equal to or less than 200 nm, such as from 50 to 200 nm, to achieve optimal delivery efficiency to the target cell. |

The ability of viral polypeptides to bind to nucleic acids can be determined in vitro using techniques such as gel electrophoresis, including gel retardation assays (see Materials and Methods and Results) and electrophoretic strip mobility motions, ethidium bromide exclusion assays, and affinity. chromatography (for example, using single stranded or double stranded DNA coupled cellulose).

For example, the ability of viral polypeptides to condense nucleic acids can be determined by circular dichroic (CD) spectroscopy (see, e.g., Sato and Hosokawa, 1984, J. Biol. Chem. 95: 1031-1039).

Viral polypeptides or homologues or derivatives thereof will generally contain a number of positively charged amino acid residues at physiological pH (such as pH 7.4). Preferably, the total net charge of the viral polypeptide at physiological pH is positive. It is particularly preferred that the charge: amino acid ratio is at least +0.3, preferably at least +0.4, +0.5 or +0.6.

It is preferred that the viral polypeptides, or homologues or derivatives thereof, contain arginine residues rather than lysine residues or a mixture of both. It is also particularly preferred that the viral polypeptides or homologues or derivatives thereof comprise one or more histidine residues, preferably two or more histidine residues. In addition, viral polypeptides or homologues or derivatives thereof will typically contain a number of highly hydrophobic residues such as alanine, for example two or more hydrophobic residues.

It will be appreciated that the amino acid sequences for use in the present invention are not limited to naturally occurring viral nucleic acid packaging polypeptides, but also include homologous sequences obtained from any source, for example, related viral / bacterial proteins, cell homologues and synthetic peptides as well as variants thereof. or derivatives, such as fragments,

In the context of the present invention, a homologous sequence is defined to include an amino acid sequence that is at least 60, 70, 80 or 90% identical, preferably at least 95 or 98% identical at the amino acid level along at least 10, preferably at least 20, 30, 40 or 50 amino acids, with a viral core polypeptide, for example, the Mul sequence shown as .... SEQ ID NO. 1. Homology should typically be compared with respect to those regions of the sequence that are known to be necessary for nucleic acid binding, rather than with respect to surrounding expendable sequences. Although homology can also be compared for similarity (i.e., amino acid residues with similar chemical properties / functions), in the context of the present invention it is preferred to express homology in terms of sequence identity.

Homology comparisons can be performed to the naked eye, or more usually using readily available sequence comparison programs. These commercially available computer programs can calculate the percent homology between two or more sequences.

Percent homology can be calculated along contiguous sequences, ie one sequence is aligned next to the other sequence, and each amino acid in one sequence is directly compared to the corresponding amino acid in the other sequence, each by individual residues. This is called "ungapped" comparison. Such uninterrupted comparisons are typically performed only along a relatively small number of residues (e.g., less than 50 consecutive amino acids).

-5GB 298353 B6

Although this method is very simple and reliable, it does not take into account that, for example, in an otherwise identical sequence pair, one insertion or deletion will result in a misalignment of the following amino acid residues, potentially resulting in a significant decrease in percent homology when overall alignment is performed. Thus, most sequence comparison methods are designed to provide optimal alignments that take into account possible insertions and deletions without unduly penalizing the overall homology rating. This is accomplished by inserting gaps in sequence comparison by attesting to achieve maximum local homology.

However, these more complex methods assign certain gap penalties to each gap that occurs in the comparison, so that for the same number of identical amino acids, a better ranking is achieved when comparing sequences with as few gaps as possible - reflecting a higher relationship between the two compared sequences - than sequences containing a large number of gaps. Typically, 'afftne gap costs' are used to attribute a relatively large rating to the existence of a gap and smaller penalty points for each subsequent gap gap. This is the most commonly used gap rating system. Of course, high gap penalty points will provide optimized comparisons with fewer gaps. Most comparison programs allow for modification of gap penalty points. However, when such software is used for sequence comparison, it is preferable to use default values. For example, if the GCG Wisconsin Bestfit package program (see below) is used, the default gap penalty points for amino acid sequences are -12 for the gap and -4 for each extension.

Therefore, calculating the maximum percentage of homology first requires the creation of an optimal comparison, taking into account the gap penalty points. A suitable computer program for making such a comparison is the GCG Wisconsin Bestfit package program (University of Wisconsin, USA; Devereux et al., 1984, Nucleic Acids Research 12: 387). Examples of other programs that can be used for sequence comparison include, but are not limited to, the BLAST package program (see Ausubel et al., 1999 ibid. - Chapter 18), FASTA (Atschul et al., 1990, J. Mol. Biol., 403 410) and a set of GENEWORKS benchmarking tools. Both BLAST and FASTA are available for offline and online scanning (see Ausubel et al., 1999 ibid., Pp. 7 - 58 to 7 - 60). However, it is advantageous to use the GCG Bestfit program.

Although the final percentage of homology can be measured in terms of identity, the comparison process itself is not typically based on comparing all or nothing pairs. Instead, a certain similarity matrix is generally used, which assigns an assessment to each pair comparison based on chemical similarity or evolutionary distance. An example of such a commonly used matrix is the BLOSUM62 matrix - a preset matrix for the BLAST program family. GCG Wisconsin programs generally use either generally known default values or individual symbol comparison tables, if supplied (see user manual for more details). It is advantageous to use generally known preset values for the GCG program family or, in the case of other software, a preset matrix such as BLOSUM62.

Once the optimum alignment program has been established, the percent homology, preferably percent sequence identity, can be calculated. The program typically does this as part of a sequence comparison and provides a numerical result.

The term "derivative" relating to amino acid sequences used in the present invention includes any substitution, variation, modification, substitution, deletion or addition of one (or more) amino acid from or to a sequence to provide the resulting amino acid sequence with nucleic acid binding ability and condensation activity , preferably with at least the same activity as the unmodified polypeptides.

Viral polypeptides may be modified for use in the present invention. Typically, modifications are made that retain nucleic acid binding and condensation properties of the sequence. For example, amino acid substitutions may consist of 1, 2 or 3 to 10, 20 or 30 substitutions, provided that the modified sequence retains the ability to bind

-6E 298353 B6 nucleic acids and condensation properties. Amino acid substitutions may include the use of non-natural analogs, for example, to increase the plasma half-life of a therapeutically administered polypeptide.

In particular, it may be desirable to create amino acid substitutions to increase the overall positive charge at physiological pH of the viral packaging polypeptide occurring in nature. Positively charged amino acids include arginine, lysine and histidine. Arginine has the highest positive charge of the naturally occurring amino acids and is therefore particularly preferred.

Aliphatic amino acid Nonpolar GAP ILV Polar - uncharged CSTM NQ Polar - charged DE KR Aromatic amino acid HFWY

Conservative substitutions may be made, for example, based on the above table. Amino acids in the same region in the second column and preferably in the same row in the third column may be replaced.

Polypeptides for use in the invention may be produced by recombinant methods, for example as described below. However, they can also be produced synthetically using technologies well known to those skilled in the art, such as solid phase synthesis. Polypeptides for use in the invention may also be produced as fusion proteins, for example, to facilitate extraction and purification. Examples of fusion protein partner portions include glutathione-S-transferase (GST), 6 x His, GAL4 (DNA binding and / or transcriptional activation domains), and β-galactosidase. It may also be desirable to add a proteolytic enzyme cleavage site between the fusion protein partner portion and the selected protein sequence to allow removal of the fusion protein sequences. Preferably, the fusion protein partner portion will not limit the biological activity of the selected protein sequence.

The polypeptides for use in the present invention may be in substantially isolated form. It will be appreciated that the polypeptides may be mixed with carriers or diluents that will not interfere with the intended use of the polypeptide and will still be considered substantially isolated. The polypeptides may also be in substantially purified form, in which case, in general, the polypeptides for use in the invention comprise more than 90%, for example 95%, 98% or 99% of the proteins in the preparation.

2. Polypeptides comprising nuclear localization sequences

In a preferred embodiment, the delivery vector / complex of the invention additionally comprises a polypeptide comprising a nuclear localization sequence (NLS). Nuclear localization sequences are well known in the art (see, for example, Dingwall and Laskey, 1991, Trends. Biochem. Sci. 16: 478-481). However, it is particularly advantageous to use the nuclear localization sequences of the adenoviral pV protein. The nuclear localization sequence of the pV protein has the sequence

RPRRRATTRRRTTTGTRRRRRRR (SEQ ID No. 2), which corresponds to amino acids 315-337 (D. Matthews). Another nuclear localization sequence is present at the N-terminus (KPRKLKRVKKKKK - SEQ ID No. 3), although the C-terminal nuclear localization sequence is preferred.

The nuclear localization sequence may be present on a different polypeptide molecule than the packaging polypeptide or as part of the same polypeptide chain, for example, in the form of a fusion protein.

B. Selected nucleic acid sequences

The selected nucleic acid sequences of interest (NOI) are to be delivered to the cells using the delivery vector or complex of the invention and may be DNA or RNA. They may be single-stranded or double-stranded. They may also be polynucleotides that comprise synthetic or modified nucleotides. A number of different types of oligonucleotide modifications are known in the art. These include methylphosphonate and phosphorothioate backbones, adding acridine or polylysine chains to the 3 'and / or 5' ends of the molecule. For the purposes of the present invention, it will be understood that the polynucleotides described herein may be modified by any method available in the art. These modifications can be made to increase the in vivo activity or in vivo half-life of the selected nucleic acid sequences.

The selected nucleic acid sequence typically comprises a heterologous gene. The term "heterologous gene" includes any gene. The heterologous gene may be any allelic variant of the wild-type gene or may be a mutant gene. The term "gene" is intended to include nucleic acid sequences which are capable of at least transcription. Thus, sequences coding for mRNA, tRNA, and rRNA, as well as positive (antisense) constructs, are included in this definition. The nucleic acids may be, for example, ribonucleic acid (RNA) or deoxyribonucleic acid (DNA) or analogs thereof. The mRNA coding sequences will optionally contain some or all of the 5 'and / or 3' transcribed but non-translated flanking sequences that are naturally or otherwise associated with the translated coding sequence. They may optionally further comprise associated transcriptional control sequences that are normally associated with transcribed sequences, such as transcription termination signals, polyadenylation sites, and enhancers in the transcription direction.

Preferably, the transcribed heterologous gene sequence is operably linked to a control sequence allowing expression of the heterologous gene in mammalian cells, preferably neuronal cells, such as cells of the central and peripheral nervous system, cancer cells, or epithelial cells. The term " operably linked " refers to a state in which the components described are in a relationship that allows them to function as intended. The control sequence "operably linked" to the coding sequence is linked in such a way that expression of the coding sequence is achieved under conditions compatible with the control sequence.

The control sequence comprises a promoter allowing expression of the heterologous gene and a transcription termination signal. The promoter is selected from promoters functional in mammalian, preferably human, cells. The promoter may be derived from promoter sequences of eukaryotic genes. For example, it may be a promoter derived from the genome of a cell in which the heterologous gene is to be expressed, preferably a mammalian central or peripheral nervous system cell. For eukaryotic promoters, they may be promoters operating in a general manner (such as β-actin, tubulin promoters) or alternatively in a tissue-specific manner (such as pyruvate kinase gene promoters). They may also be promoters that respond to specific stimuli, for example promoters that bind to steroid hormone receptors. Viral promoters, such as the promoter (MMLV LTR, Moloney murine leukaemia virus long terminal repeat) or herpes virus gene promoters, may also be used.

It may also be advantageous if the promoters are inducible so that the expression level of the heterologous gene can be controlled over the life of the cells. Inducible means that expression levels occurring using the promoter can be controlled.

-8EN 298353 B6

In addition, any of these promoters may be modified by the addition of additional control sequences, such as enhancer sequences. Chimeric promoters comprising sequence elements from two or more different promoters described above may also be used. The use of LCR (locus control regions) may also be desirable.

The heterologous gene will typically encode a polypeptide for therapeutic use. According to the present invention, suitable selected nucleic acid sequences include those having therapeutic and / or diagnostic uses, including but not limited to: sequences encoding cytokines, chemokines, hormones, antibodies, genetically engineered immunoglobulin-like molecules, single-chain antibodies fusion proteins, enzymes, helper immune stimulating molecules, immunomodulatory molecules, antisense RNA, transdominant negative mutant of the target protein, toxin, conditional toxin, antigen, tumor suppressor and growth factor protein, membrane proteins, vascular-acting proteins and peptides, antiviral proteins and ribozymes and their derivatives (e.g., an associated reporter group).

Examples of polypeptides for therapeutic use include neurotrophic factors such as nerve growth factor (NGF), ciliary neurotrophic factor (CNTF), brain neurotrophic factor (BNTF), and neurotrophins (such as NT-3, NT-4/5) that have the potential as therapeutic agents for the treatment of neurological diseases such as Parkinson's disease.

Suitable selected nucleic acid sequences for use in the treatment or prevention of cancer include selected nucleic acid sequences encoding proteins that: destroy a target cell (e.g., a ribosomal toxin), act as: tumor suppressants (such as wild-type p53 protein); activators of anti-tumor immune mechanisms (such as cytokines, helper stimulatory molecules, and immunoglobulins); angiogenesis inhibitors; or that provide increased sensitivity to drugs (such as precursor activating enzymes); indirectly stimulate target cell destruction by natural effector cells (for example, a potent antigen to stimulate the immune system or convert a precursor substance to a toxic substance that destroys the target cell (for example, a precursor activating enzyme). Coded proteins could also destroy cells adjacent to tumor cells (for example protein antitumor antibody - ribosomal toxin), indirectly stimulate destruction of adjacent tumor cells (for example, cytokines to stimulate the immune system or proteins that promote coagulation that cause local closure of blood vessels) or convert a precursor substance to a toxic substance that destroys adjacent tumor cells (for example selected nucleic acid sequences may be those that encode positive (antisense) transcripts or ribozymes that interfere with cell expression For example, they may be associated with transient or pathogenic genes, for example, expressing cellular tumor persistence genes (e.g., against different myc transcripts in Burkitts lymphoma or against bcr-abl transcripts in chronic myeloid leukemia. The use of combinations of these selected nucleic acid sequences is also contemplated.

Instead, or as in the case of selective expression in target tissues, the selected nucleic acid sequences may encode a precursor activating enzyme or enzymes that have no significant effect or no adverse effect as long as the individual is treated with one or more precursors on which the enzyme or enzymes act. In the presence of an active nucleic acid sequence of choice, treatment of an individual with a suitable precursor results in an enhanced reduction in tumor growth or survival.

The precursor activating enzyme may be delivered to a tumor site for the treatment of cancer. In any case, a suitable precursor is used in the treatment of a patient in combination with a suitable precursor activating enzyme. A suitable precursor is administered with the vector. Examples of precursors include: etoposide phosphate (with alkaline phosphatase); 5-fluorocytosine (with cytosine deaminase); doxorubicin-N-phydroxy-phenoxyacetamide (with penicillin-V-amidase); para-N-bis (2-chloroethyl) aminobenzoyl glutamate (with carboxypeptidase G2); carbamates of cephalosporin with nitrogen mustard (with β

-9EN 298353 B6 lactamase); SR4233 (with P450 reductase); ganciclovir (with HSV thymidine kinase); nitrogen mustard precursors with nitroreductase and cyclophosphamide (with P450).

Examples of suitable prodrug-activating enzymes for use in the invention include thymidine phosphorylase that activates the 5-fluorouracil precursors capetetabine and furtulon; herpes simplex virus thymidine kinase that activates ganciclovir; and cytochrome P450, which activates a precursor such as cyclophosphamide to a DNA damaging agent; and a cytosine deaminase that activates 5-fluorocytosine. Preferably, an enzyme of human origin is used,

The nucleic acid sequences of choice may also encode antigenic polypeptides for use as vaccines. These antigenic polypeptides are preferably derived from a pathogenic organism, for example, bacteria or viruses. Examples of such antigenic polypeptides include hepatitis C virus antigens, hepatitis B virus surface or nuclear antigens, HIV antigens, pertussis toxin, chole toxin, or diphtheria toxin.

The nucleic acid sequences of choice may also contain marker genes (e.g., encoding β-galactosidase or a green fluorescent protein) or genes whose products direct the expression of other genes (e.g., transcription control factors).

If the disease is caused by gene defects, selected nucleic acid sequences that encode the fully functional allele of the gene may be administered, as is the case with cystic fibrosis. So far, the molecular basis for a variety of genetic disorders has been identified, and wild-type functional sequences have been cloned. It may be desirable to include flanking sequences of a selected nucleic acid sequence that are homologous to the corresponding flanking sequences in the genome to allow replacement of the defective gene by homologous recombination.

Gene therapy and other therapeutic uses may also require the administration of multiple genes. Multiple gene expression may be beneficial for the treatment of a variety of conditions. Since there is no restriction on the size of the selected nucleic acid sequence that can be inserted into the delivery vector or complex of the invention, it should be possible to direct multiple genes to the cells simultaneously.

C. Cationic lipids

A variety of cationic lipids are known in the art, see for example WO 95/02698, the contents of which are hereby incorporated by reference, some of which are reproduced below. Examples of cationic lipid structures useful in the present invention are shown in Table 1 of WO 95/02698. In general, any cationic lipid, either monovalent or polyvalent, may be used in the vaccines and methods of the present invention. In general, polyvalent cationic lipids are preferred. Cationic lipids include saturated and unsaturated alkyl and alicyclic ethers and esters of amines, amides or derivatives thereof. The straight and branched chain alkyl and alkene groups of the cationic lipids may contain from 1 to about 25 carbon atoms. Preferred straight or branched chain alkyl and alkene groups contain 6 or more carbon atoms. Alicyclic groups may contain from about 6 to 30 carbon atoms. Preferred alicyclic groups include cholesterol and other steroid groups. Cationic lipids can be prepared with a variety of counterions (anions) such as, but not limited to, chloride, bromide, iodide, fluoride, acetate, trifluoroacetate, sulfate, nitrite, and nitrate.

A well known cationic lipid is N- [1- (2,3-dioleyloxy) propyl] -N, N, N-trimethylammonium chloride (DOTMA).

DOTMA and the analogous diester DOTAP (1,2-bis (oleoyloxy) -3- (trimethylammonium) propane) are commercially available. Other cationic lipids structurally related to DOTMA are described in U.S. Patent 4,897,355, which is hereby incorporated by reference.

-10GB 298353 B6

Another useful class of cationic lipids related to DOTMA and DOTAP include substances commonly called DORI ethers and / or DORI esters. DORI lipids differ from DOTMA and DOTAP in that only one of the methyl groups of the trimethylammonium group is replaced by a hydroxyethyl group. The oloyl groups of the DORI lipids may be replaced by other alkyl or alkene groups such as palmitoyl or stearoyl groups. The hydroxyl group of the DORI-type lipids can be used as a site for further functionalization, for example, for esterification to amines such as carboxyspermine.

Other cationic lipids that can be used in delivery vectors or complexes of the invention include the compounds described in WO 91/15501 as useful for transfecting cells.

In the context of the present invention may also be used cationic sterol derivatives such as 3? [N- (N ', ^N' -dimethylaminoethan) carbamoyl] cholesterol (DC-Chol) in which cholesterol is linked to a trialkylammonium group. DC-Chol is intended to provide more efficient transfection at lower toxicity than DOTMA-containing liposomes in some cell lines. DC-Chol polyamine variants, such as those described in WO 97/45442, can also be used.

Carboxyspermine-containing ionic lipids may also be used in delivery vectors or complexes of the invention. EP-A-304111 discloses carboxyspermine-containing cationic lipids including 5-carboxyspermylglycindioctadecylamide (DOGS) and dipalmitoyl phosphatidylethanolamine-5-carboxyspermylamide (DPPES). Other cationic lipids can be obtained by replacing octadecyl and palmitoyl groups DOGS and DPPES with other alkyl or alkene groups.

In the delivery vectors or complexes of the invention, the cationic lipids may optionally be combined with non-cationic helper lipids, preferably neutral lipids, to form liposomes or lipid aggregates. Neutral lipids useful in the present invention include many others: lecithins; phosphatidylethanolamines such as DOPE (dioleoylphosphatidylethanolamine), POPE (palmitoyloleoylphosphatidylethanolamine) and DSPE (distearoylphosphatidylethanolamine); phosphatidylcholine; phosphatidylcholines such as DOPC (dioleoylphosphatidylcholine), DPPC (dipalmitoylphosphatidylcholine) POPC (palmitoyloleoylphosphatidylcholine) and DSPC (distearoylphosphatidylcholine); phosphatidylglycerol; phosphatidylglycerols such as DOPG (dioleoylphosphatidylglycerol), DPPG (dipalmitoylphosphatidylglycerol) and DSPG (distearoylphosphatidylglycerol); phosphatidylserines such as dioleoyl- or dipalmitoylphosphatidylserine; diphosphatidesglycerols; fatty acid esters; glycerol esters; sphingolipids; cardolipin; cerebrosides; and ceramides; and mixtures thereof. Neutral lipids also include cholesterol and other 3DOH-sterols.

Optionally, one or more amphiphilic compounds may be included in the delivery vectors or complexes of the invention to modify surface properties. Amphiphilic compounds useful in the present invention include, but are not limited to, neoglycolipids such as GLU4 and GLU7 shown in Figure 22, polyethylene glycolipids such as N- (n-methoxy (polyoxyethylene) oxycarbonyl) phosphatidylethanolamine, N-mono-methoxy (polyoxyethylene) succinylphosphatidylethleneaminol, and poly; nonionic detergents such as alkyl glycosides, alkylmethylglucamides, sucrose esters, alkyl polyglycerol ethers, alkyl polyoxyethylene ethers and alkylsorbitanoxyethylene ethers and steroid oxyethylene ethers; block copolymers such as block copolymers of polyoxyethylene polyoxy propylene.

In one embodiment, the cationic lipid of the present invention is modified with a sugar group or a polyethylene glycol (PEG) group. In another embodiment, the complex of the invention further comprises a compound capable of acting as a cationic lipid, wherein the compound comprises a linked cholesterol group via an amino group, a sugar group, or a polyethylene glycol group. As shown in the examples, the inventors have found that sugars / PEG modified cationic lipids are particularly preferred. Another embodiment of the present invention

Thus, it provides a compound capable of acting as a cationic lipid, wherein the compound comprises a cholesterol group linked through an amine group, a sugar group, or a polyethylene glycol group. Preferably, the compound contains from 1 to 7 sugar groups or polyethylene glycol groups. The compound may comprise a mixture of sugar groups and polyethylene glycol groups. The sugar moiety is preferably glucose or D-glucose or a moiety derived therefrom.

D. Complexes cationic lipid / selected nucleic acid sequence / packaging polypeptide

The delivery vector / complex of the present invention is typically prepared by first contacting the packaging polypeptide and the selected nucleic acid sequence in a sterile tube for about 10 minutes at room temperature, resulting in the formation of a fused polypeptide / selected nucleic acid sequence. A common method is to apply the nucleic acid and protein side by side into a test tube so that they are not in contact, and initiate mixing by adding several hundred microliters of a liquid carrier such as a pharmaceutically acceptable carrier, excipient or diluent.

Another and preferred method of preparing the delivery vector or complex of the present invention is by contacting the packaging polypeptide and the selected nucleic acid sequence with constant shaking.

The ratio of the selected nucleic acid sequence to the polypeptides is at least 1: 1, preferably from 1: 1 to 2: 1, more preferably from 1.4: 1 to 1.9: 1, even more preferably 1.5: 1 to 1.8: The inventors have found that a ratio of selected nucleic acid sequence to polypeptides of about 1: 0.6 (~ 1.7: 1) is particularly preferred. In some embodiments, the ratio of polypeptides to the selected nucleic acid sequence is typically from 0.2 to 1.5, preferably from 0.3 to 1.2 (w / w), even more preferably from 0.5 to 0.7 . In other embodiments, the typical ratio of polypeptides to selected nucleic acid sequence is at least 10: 1, or at least 20: 1 (w / w). However, the optimum ratio may depend on the charge: amino acid ratio of the packaging polypeptide. In general, the lower the charge: amino acid ratio, the higher the polypeptide: nucleic acid sequence ratio used.

Cationic lipids are further added to the complex. The cationic lipids may in one embodiment be part of a preformed liposome comprising two or more lipid components such as DC-Chol and DOPE. The cationic lipids are typically incubated with the polypeptide / selected nucleic acid sequence complex for at least 20 min at room temperature. Another and preferred method of adding cationic lipids is by adding in the form of a suspension of cationic liposomes. This finished complex can be stored at about -80 ° C with the addition of 10% sucrose (w / v) until use. The amount of liposome relative to the selected nucleic acid sequence is typically of the order of from 3: 1 to 20: 1, preferably from 6: 1 to 15: 1, more preferably from 8: 1 to 14: 1. liposome: a selected nucleic acid sequence of 12: 1. In other embodiments, the ratio of liposome: a selected nucleic acid sequence is typically of the order of 2: 1 to 10: 1, or from 3: 1 to 6: 1. When cationic lipids are used with neutral lipids, this ratio is typically of the order of 1: 1.

In a very preferred embodiment, the liposome: NO1 ratio is

30-20: 1 preferably 8-14: 1 more preferably ~ 12: 1 polypeptide

0.5-1

0.5-0.7 —0.6

The delivery vector / complex is now ready for use. While it is preferable to mix the various components in the above order, it is possible to combine the components in any order. If additional polypeptide components are to be added, they may be added at any stage, but preferably together with the packaging polypeptide.

-12GB 298353 B6

It may be desirable to add additional components to the vectors / complexes, for example, cell surface receptor binding ligands to obtain vectors / complexes with a degree of selectivity for a particular cell type. Ligands include peptides, glycoproteins, oligosaccharides, lectins, and counterparts and fragments thereof,

E. Administration

The delivery vector / complex of the invention is preferably combined with a pharmaceutically acceptable carrier or diluent to form a pharmaceutical composition (which may be for human or veterinary use). Suitable carriers and diluents include saline solutions, for example phosphate buffered saline. The composition of the invention may be administered by direct injection. The composition may be formulated for parenteral, intramuscular, intravenous, subcutaneous, intraocular or transdermal administration or inhalation. Typically, each selected nucleic acid sequence may be administered at a dose of from 10 ng to 10 pg / kg body weight, 15 preferably from 0.1 to 10 pg / kg, more preferably 0.1 to 1 pg / kg body weight.

Alternatively, transfection of the patient's cells may be performed ex vivo by removing the patient's tissue, transfecting using the delivery vector / complex of the invention, and then reimplanting the transfected tissue.

Routes of administration and dosages used are provided for guidance only, as one skilled in the art will be able to readily determine the optimal mode of administration and dosage for any particular patient and condition.

F. Use

The delivery vectors / complexes of the present invention can be used to efficiently transfect eukaryotic cells, particularly mammalian cells, with selected nucleic acid sequences. Delivery vectors / complexes have been shown to be particularly effective compared to prior art compositions in transfecting neuronal cells. This is of particular relevance for (i) research where nerve cells are used and (ii) clinical applications where the introduction of selected nucleic acid sequences into cells of the central or peripheral nervous system of a human or animal is required. More generally, delivery vectors / complexes of the present invention can be used in a variety of applications to deliver selected nucleic acid sequences, such as gene therapy, delivery of DNA vaccines, and in vitro transfection studies.

Examples of diseases that may be suitable for treatment using the complexes / vectors of the invention include diseases of the peripheral or central nervous system, such as neurodegenerative diseases, and nerve tissue damage due to injury / injury (including stroke). Neurodegenerative diseases include motor neuron diseases, several hereditary diseases such as familial dysautonomy and infant spinal muscular atrophy, and late-onset neurodegenerative diseases such as Parkinson's and Alzheimer's disease.

The delivery vectors / complexes of the invention can also be used to deliver therapeutic genes to patients suffering from a malignant disease. Examples of malignant diseases that may be suitable for treatment are breast, cervical, colon, rectum, endometrium, kidney, lung, ovary, pancreas, prostate, skin, stomach, bladder, central nervous system, esophagus, head or throat, liver, testes, thymus or thyroid. Malignant diseases of blood cells, bone marrow cells, B-lymphocytes, T-lymphocytes, lymphocyte precursors or myeloid cell precursors may also be suitable for treatment.

The tumor may be a solid tumor or a non-solid tumor and may be a primary tumor or a disseminated metastatic (secondary) tumor. Non-solid tumors include myeloma; leukemia (acute or chronic, lymphocyte or myelocytic) such as acute myeloblastic, acute promyelocytic, acute myelomonocytic, acute monocytic, and eryhtroleukemia;

- 13GB 298353 B6 and a lymphorrh such as Hodgkin's, other than Hodgkin's and Burkitt's lymphoma. Solid tumors include carcinoma, colon carcinoma, small lung cell carcinoma, non-small lung cell carcinoma, adenocarcinoma, melanoma, basal or squamous cell carcinoma, mesothelioma, adenocarcinoma, neuroblastoma, glioma, astrocytoma, medulloblastarcoma, retinoblastoma, sinoma rhabdomyosarcoma, fibrosarcoma, osteogenic sarcoma, hepatoma and seminoma.

Other diseases include diseases caused by mutations, inherited or somatic, in normal cell genes such as cystic fibrosis, various thaasemias and the like.

Another area of interest includes the treatment of immune-related diseases such as organ transplant rejection and autoimmune diseases. The spectrum of autoimmune diseases ranges from organ specific diseases (such as thyroiditis, insulitis, multiple sclerosis, iridocyclitis, uveitis, orchitis, hepatitis, Addison's disease, myasthenia gravis) to systemic diseases such as rheumatoid arthritis and other rheumatic diseases or lupus erythema. Other diseases include excessive reactivity of the immune system, such as allergic reactions, especially those associated with histamine production and asthma.

The present invention will now be illustrated by the following non-limiting examples.

BRIEF DESCRIPTION OF THE DRAWINGS

Giant. 1 shows a plate;

Fig. 2 shows a graph;

Fig. 3 shows a plate;

Fig. 4 shows a graph;

Fig. 5 shows a graph;

Fig. 6 shows a graph;

Fig. 7 shows a graph;

Fig. 8 shows a graph;

Figure 9 shows structures;

Fig. 10 shows a graph;

Fig. 11 shows a graph;

Fig. 12 shows a graph;

Fig. 13 shows a graph;

Fig. 14 shows a graph;

Fig. 15 shows a graph;

Fig. 16 shows a plate;

Fig. 17 shows a graph;

Fig. 18 shows a graph;

Fig. 19 shows a structure;

Figure 20 shows a reaction scheme;

Fig. 21 shows a reaction scheme;

Figure 22 shows structures;

Fig. 23 shows the principle of micelle incorporation;

Fig. 24 shows a graph; and Fig. 25 shows a graph.

-14GB 298353 B6

Detailed Description of Figures 1 to 6

Giant. 1 - Adenoviral nuclear protein Mul is more effective in plasmid DNA binding than polyomavirus Vpl nuclear protein

A) BSA has no effect on pDNA electrophoretic mobility, 1 µg pCMVp was incubated with 0 µg (lane 2), 5 µg (lane 3), 10 pg (lane 4), 15 pg (lane 5), 20 pg (lane) 6), 25 µg (lane 7) and 30 µg (lane 8) of BSA for 10 min at room temperature in 1 x HBS buffer. The samples were then analyzed on a 1% agarose gel for altered motility. No change in electrophoretic mobility caused by BSA was detected.

B) Unlike BSA, Mul interfered dramatically with pDNA motility. pCMVp (1 µg) was incubated with 0.25 µg (lane 2), 0.5 µg (lane 3), 1 µg (lane 4), 2 µg (lane 4), 4 µg (lane 6), 6 µg (lane 3). lane 7) and 0 µg (lane 8) of recombinant Mul peptide as in case A. While protein to pDNA ratios of 0.25 (w / w) (lane 2) did not affect migration of the relaxed form of pCMVp (upper lane), moderate observed deceleration of the supercoiled pDNA (lower band). When ratios of 0.5 (w / w) or higher were used, the migration of both forms of pDNA was significantly slowed.

C) The polyomavirus Vp1 protein had a much lower potency in preventing pDNA migration. pCMV (1 pg) was incubated with 2 pg (lane 2), 4 pg (lane 3), 6 pg (lane 4), 8 pg (lane), 16 pg (lane 6), 32 pg (lane 7) and 0 pg (lane 8) Vpl. Only ratios of 6 or greater (protein: pDNA, w / w) caused a significant retardation of the supercoiled form of pDNA (lane 6, lower band). Also, up to a 32 (w / w) ratio, no effect on relaxed pDNA (lane 7, upper lane) was observed. For all gels, lane 1 corresponds to 1 kb DNA (BRL).

Giant. 2 - Activity of β-galactosidase in ND7 cells transfected with pDNA-Mulkationic liposome complexes

ND7 cells were seeded at a density of 5 x 10 ⁴ cells / well in 24-well culture dishes 24 hours prior to transfection. Immediately prior to transfection, cells were washed with serum-free medium. The complexes were formed by incubating pCMVp with Mul before adding the cationic liposome DC-Chol / DOPE. In each case, a complex of 1 µg of pCMVβ was formed with 0.6, 6, 12 and 21 µg of the Mul peptide. Each of these combinations was then formulated to complex with 3, 4 and 6 µg DC-Chol / DOPE. ND7 cells were exposed to the transfection complexes for 2 hours, then maintained at 37 ° C, 5% CO _{2 for an} additional 24 hours before harvesting and processing to perform an β-galactosidase enzyme assay. Values are mean ± SD, n = 3.

Giant. 3-Mul enhances the efficiency of cationic liposome-mediated transfection in the neuronal ND7 cell line

ND7 neurons were seeded into 24-well culture dishes at a density of 4 x 10 ⁴ cells / well and allowed to grow for 24 hours. The undifferentiated ND7 neurons were then transfected with either pCMVp (A) alone, pCMVp complexed with DC-Chol / DOPE (1). (3) (w / w), (B) or pCMVβ complexed with Mul and DC-Chol / DOPE (1/12/6) (C). 48 hours later, cells were fixed and processed for histochemical detection of X-Gal. As seen in section C, the addition of Mul to the complex at an optimal ratio significantly increases the number of X-Gal positive cells (blue staining).

Giant. 4 - Mul has much higher potency in enhancing cationic liposome-mediated transfections in ND7 cells than Vpl

Plasmid DNA of pCMVp was complexed with various amounts of polycationic peptide and then mixed with cationic liposomes at a ratio of 1: 3 (pCMVp: liposomes; w / w). After rapid washing in serum-free medium, ND7 cells were exposed

- 15GB 298353 B6 liposome-polycation-liposome complexes for 2 hours and then returned to serum-containing medium. 48 hours later, the cells were harvested and processed for the β-galactosidase enzyme assay. Each configuration was tested in triplicate and each experiment was repeated three times. Values mean mean + SD.

Giant. 5 - Mul enhancers of DC-Chol / DOPE transfection in COS-7 cells

COS cells were seeded at 60-80% confluency in 24-well culture dishes 24 hours prior to transfection. Immediately prior to transfection, cells were washed in serum-free medium. Incubation of pCMVp with Mul before addition of complexes formed from cationic liposome

DC-Chol / DOPE capable of cell transfection. In each case, a complex of 1 gg of pCMVp was formed with 12 µg of Mul peptide, which was found to be optimal for ND7 cells. PCMVp: Mull complexes were then mixed with 3, 4 and 6 gg DC-Chol / DOPE. COS cells were exposed to the transfection complexes for 2 hours and then maintained at 37 ° C, 5% CO 2 for an additional 24 hours before harvesting and processing for the β-galactosidase enzyme assay. Numeric values mean mean ± SD, n = 3.

Giant. 6 - Transfection efficiency in differentiated cells of ND7 pCMVβ-Mulcationic liposome complexes

ND7 cells were seeded into 24-well culture plates at a density of 4 x 10 ⁴ cells / well in normal growth medium (+ serum). 24 hours later, the medium was replaced with differentiation medium and the cells were cultured for another 24 hours. Three differentiation media were used: serum free (- serum), normal growth medium plus 1 mM cAMP (cAMP), or reduced serum (0.5%). plus 1 mM cAMP and 50 ng / ml nerve growth factor (NGF). Cells were then transfected with pCMVβ complexed with either DC-Chol / DOPE alone or Mul plus DC-Chol / DOPE. 48 hours later, cells were fixed and processed for histochemical X-Gal determination, and the percentage of positive cells was determined. In all cases, the presence of Mul increased the number of positive cells. Interestingly, the number of transfected cells was increased in cells grown in the presence of cAMP both with and without Mul.

DETAILED DESCRIPTION OF THE INVENTION

Materials and methods

Synthesis of peptides

The peptides Vp1 and Mul were synthesized on a Shimadzu PSSM-8 solid phase synthesizer using a 5-fold excess of (9-fluorenyl) methoxycarbonyl (Fmoc) -protected L-amino acids (Novabiochem) and FastMoc ™ 2- (1H-benzoztriazol-1-yl) reagents. 1 -1,3,3-tetramethyluronium hexafluorophosphate / hydroxybenztriazole (HBTU / HOBt) (Advanced Chemtech Europe) as an amide group coupling agent. After resin cleavage and deprotection, desalting was performed by gel filtration on a P2 Biogel column (2 x 28 cm; Biorad) coupled to an FPLC system (Amersham Pharmacia Biotech UK) with 0.1% aqueous TFA as eluent at a flow rate of 0.5 to 0.75 ml. Final min reverse phase preparative purification was performed on a Vydac column (C18, 5 μιη, 2 x 25 cm; Hichrom) coupled to a Gilson HPLC system (Anachem). The peptides were eluted at 5 ml / min with a linear gradient of acetonitrile in 0.1% aqueous TFA and the elution was monitored at 220-230 nm.

The Vp1 peptide was prepared using acid-labile resin β-Pro-2-chlorotrityl super acid labile resin (Novabiochem) (100 mg, 1.05 mmol / g, 0.1 mmol). Extended reaction times were used to incorporate all amino acid residues from the sixth (Lys) to the N-terminal residue. After automatic removal of the N-terminal Fmoc protecting group with piperidine (20%, v / v) in dimethylformamide, the resin was isolated, washed with dimethylformamide (10 mL) and methanol (15 mL), and then dried in vacuo. The crude peptide was cleaved from the ice-cooled TFA resin (8 mL), containing phenol (7%, w / v), ethanedithiol (2%,

298353 B6 v / v), thioanisole (4%, v / v) and water (4%, v / v) (reagent known as mixture A), and then precipitated with ice-methyl tert- butyl ether (MTBE) (30ml). The precipitated material was then desalted and the crude peptide mixture was purified by reverse phase HPLC. After elution, fractions containing the desired peptide (eluting with acetonitrile 68.5% v / v) were combined and lyophilized to give the peptide as a white powder. Total yield: 32 mg (15 pmol, 15%); MS (MALDI-TOF) C 5 H _{8 L5]} N ₂₆ O ₂₆ S 3: [M + H] ⁺ calcd 2049.5, found 2050.2. The sequence was confirmed by amino acid composition and sequence analysis. HPLC showed > 95% homogeneity.

The Mul peptide was made using Gly-Wang resin (Novabiochem) (40 mg, 0.67 mmol / g, 0.03 mmol). Normal reaction times were used. After automated removal of the N-terminal Fmoc group as above, the resin was isolated and washed with dichloromethane (20 mL) and methanol (20 mL) and then dried in vacuo. The crude peptide was cleaved from the resin using mixture A (8 mL) and precipitated with MTBE (30 mL), all as above. Finally, the crude peptide mixture was desalted and purified by reverse phase HPLC. After elution, fractions containing the desired peptide (eluting with acetonitrile 17.2%) were combined and lyophilized to give the peptide as a white powder. Total yield: 65 mg (26 pmol, 0%); MS (ES) C 8 H 10 N 4 Cl 2: [M + H] ⁺ calcd 2440.7, found 2440.6. HPLC analysis showed > 95% homogeneity.

DNA binding analysis

Purified peptides were reconstituted in sterile distilled H ₂ O at a concentration of 3 mg / ml. The peptide and pDNA were converted to complex in 20 μΐ saline with HEPES buffer (137 mM NaCl, 5 mM KCl, 0.75 mM Na ₂ HPO4, 19 mM HEPES, pH 7.4) for 20 min at room temperature. Peptide: pDNA complexes were then analyzed by agarose gel electrophoresis (1%). Control incubations for general macromolecular pDNA interactions were performed using different amounts of bovine serum albumin of molecular biology purity (Sigma).

Cell culture

ND7 cells are well characterized by a cell line derived from a fusion of neuroblastoma (NI 87g2) with sensory neurons of a newborn rat (Wood JN et al., Novel Cell-Lines Display Properties of Nociceptive Sensory Neurons. Procceding of the Royal Society of London series B-Biological sciences 1990 241, 187-194). The cell line was maintained in normal growth medium (NGM) (Leibovitz medium L-15 (BRL) supplemented with 10% fetal bovine serum (BRL) 4 g / l glucose, 4 g / l sodium bicarbonate (BRL), 100 IU / ml penicillin) (streptomycin (BRL)) at 37 ° C and 5% CO ₂ . Cells were plated in 24-well plates (Costar) at a density which gave 24% confluency after 24 hours.

ND7 cell differentiation was performed using the three methods described above (Wood JN et al., Novel Cell-Lines Display Propertis of Nociceptive Sensory Neurons. Proceeding of the Royal Society of London Series B-Biological Science 1990; 241: 187-194; Budhrammahadeo V. , Lillycrop KA Latchman DS, The Levels Of The Antagoniscite Pou Family Transcription Factors Bm-3a and Bm-3b in Neuronal Cells Are Regulated in Opposite Directions By Serum GrowthFactors. Neurosci. Leti. 1995; 185: 48-51). ND7 cells were seeded in NGM at a density of 4 x 10 ⁴ cells / well in 24-well culture dishes (Nunc). 24 hours later, the media was replaced by either a) NGM supplemented with 1 mM adenosine 3 ': 5'-cyclic monophosphate (cAMP; Sigma), or b) serum-free differentiation medium (50% Hams F12, 50% DMEM, 5 µm / ml transferrin); 250 ng / ml insulin, 0.3 μΜ sodium selenite), or (c) low serum nerve growth factor (NGF) medium (L1 5 supplemented with 2 mM glutamine, 4 g / l glucose, 4 g / l sodium bicarbonate, 10u / ml penicillin, 10g / ml streptomycin, 0.5% FCS, 1mM cAMP, 50 ng / ml NGF (Alomone Labs)). Differentiated ND7 cells were cultured in a suitable medium for 24 hours at 37 ^° C, 5% CO ₂ prior to transfection.

-17GB 298353 B6

COS-7 cells (derived from the green monkey) were grown in RPMI 1640 J (BRL) medium supplemented with 10% fetal bovine serum (BRL) and 100 IU / ml penicillin / streptomycin j (BRL). AND

Plasmid constructs9

All transfections utilized the reporter plasmid pCMVp (Clontech, Palo Alto, CA) containing the complete sequence of the E. coli β-galactosidase downstream of the early early phase promoter / enhan-9 of human cytomegalovirus (Clontech). Plasmid DNA pools were prepared by standard molecular cloning methods and purified with the Qiagen Endotoxin-$ free plasmid purification system (Qiagen, Dorking, UK).

Liposomes

DC-Chol / DOPE liposomes were prepared as described previously (Cooper RG et al., Polyamine analogues of 3 beta- [N- (N ', N'-dimethylaminoethane) carbomoyl] cholesterol (DC-Chol) as agents for gene delivery, Chemistry - a European Journal 1998; 4: 137-151; McQuilin A. et al., Optimization of liposome mediated transfection of a neuronal cell line. Neuroreport 1997; 8: 1481-1484), Briefly, 6 pmol DC-Chol and 4 DOPE (supplied at 10 mg / ml in CHCl 3) was added to freshly distilled CH ₂ Cl ₂ (5 ml) after nitrogen. To the mixture was added 5 mL of 20 mM Hepes (pH 7.8) and the mixture was sonicated for 3 min. The organic solvents were removed under reduced pressure and the obtained liposome suspension was then sonicated for an additional 3 min. Liposome preparations were stored at 4 ° C.

Transfection protocol

Since initial experiments showed that the presence of fetal bovine serum inhibited transfection of ND7 cells, serum-free differentiation medium was used for all transfections. Various amounts of DNA and liposomes were placed on the bottom of a 7 ml sterile Bijou container (Bibby Sterilin Ltd., Staffordshire, UK) with each solution not in contact with each other. DNA and liposomes were pooled by adding 400 μΐ serum-free differentiation medium and shaking gently. The DNA: liposome mixture was incubated at room temperature for 20 to 30 minutes before addition to the cells. The DNA / liposome mixture was then added to the cells and the mixture was incubated at 37 ° C, 5% CO 2 for 2 hours and then the medium was replaced with complete medium. 24-48 hours later, cells were fixed and histochemistry was processed for X-gal analysis as described in McQuillin A et al., Optimization of liposome mediated transfectison of a neuronal cell lazily. Neuroreport 1997; 8. 1481 - 1484, or harvested for β-galactosidase enzyme assays (Promega Corp.).

Cell counting was performed at 40x magnification using a Nikon Diaphot inverted microscope. Each transfection was repeated at least three times and at least three separate counts were performed for each well,

Transfection complexes containing test peptides were generated as follows. Various amounts of peptide were placed on the bottom of sterile polystyrene vials next to, but not in direct contact with, 1 µg of pCMVp, and the two solutions were mixed by adding 400 µL of serum-free NGM medium. The complexes were incubated at room temperature for 10 min and then DC-Chol / DOPE was added. The dDNA / peptide / liposome complex was then incubated at room temperature for 20 min and then added to the cells as above.

Example 1

DNA Binding Test

Mull is a polycationic peptide composed of 19 amino acids associated with the adenovirus core complex (Table 1) (Anderson C.W., Young M.E., Flint S., J., Characterization of the

-18EN 298353 B6 adenovirus 2 virion protein, mu. Virology 1989; 172: 506-512; Hosokawa K., Sung M.T., Isolation and characterization of an extremely basic protein (adenovirus type 5) Journal of Virology 1976, 17, 924-934). The present inventors compared the ability to bind Mul1 peptide DNA to the murine polyomavirus major capsid protein Vp1 by interaction with plasmid DNA in a gel retardation assay. Vpl is a 19 amino acid peptide that contains a nuclear localization signal (Chang D., Cai X., Consigli RA, Characterization of DNA binding properties of polyomavirus capsid proteins. Journal of Virology 1993; 67: 6327-6331) and contains less positively charged amino acids than the Mul peptide. It was therefore expected to have a lower ability to bind DNA.

Table 1: Protein sequences of Mul and VP1

Polypeptide Sequence Mh Charge / AA ratio Mu1 _{NH2-Met-Arg-Arg-Ala-His-His-Arg-Arg-Arg-Ala-Ser-His-Arg-Met-Arg-Gly-Gly-OH} 2440 0.63 VP1 NH? -Met-Ala-Pro-Lvs-Arq-Lvs-Ser-Glv-Val-Ser- Lys-Cys-Glu / rhr-Lys-Cys-Thr-Pro-Pro-OH 2049 0.26

The NLS sequence in VP1 peptides is cut off.

Various amounts of purified peptides were incubated at room temperature in HBS for about 10 min and then analyzed by agarose gel electrophoresis. Without the addition of peptides, supercoiled and relaxed circular plasmid DNA (pDNA) migrated as expected (Figure 1, lane 8).

Starting with the DNA: peptide ratio of Mul 1: 0.25 (w / w), the migration of plasmid DNA was slowed (Fig. 1). Plasmid DNA migration was slightly affected at 1: 0.25 (w / w), but at 1: 0.5 (w / w), plasmid DNA migration was significantly slowed and only very little material was out holes. At ratios of 2: 1 and above, pDNA was unable to migrate to the agarose gel and reduced the ability of ethidium bromide to form internal chelates with the plasmid. In contrast, for Mul, no effect on the electrophoretic mobility of plasmid DNA $ Vp1 was detected at pDNA: protein ratios up to 1: 8 (w / w) (Fig. 1). Addition of 8 µg Vp1 to 1 µg of plasmid DNA resulted in an expansion of the supercoiled pDNA band. However, no effect on the relaxed pDNA band with Vpl up to 1: 32 (w / w) pDNA: protein was observed. At this ratio, both supercoiled and relaxed pDNA bands were significantly slowed, and some DNA remained trapped in the well. No effect on electrophoretic mobility was detected when pDNA was incubated with BSA at ratios up to 1:30 (w / w) pDNA: protein (Fig. 1).

Example 2

Transfection of undifferentiated ND7 cells

The authors investigated the ability of the Mul and Vp1 peptides to enhance transfection of a neuronal cell line by cationic liposomes using a β-galactosidase reporter gene assay. ND7 cells were transfected with plasmid pCMVp complexed with different amounts of DC-Chol / DOPE peptides. The inventors have previously shown that the cationic liposome DC-Cho / DOPE is capable of effectively transfecting a neuronal derived ND7 cell line (McQuillin A. et al., Neuroreport 1997; 8: 1481).

-19GB 298353 B6

1484). In this study, it was found that optimal efficacy (> 40%) was obtained in this neuronal derived cell line using 1 µg of plasmid DNA complexed with 3 µg of DC-Chol / DOPE (McQuillin A. et al., Optimization of liposome mediated transfection of aneuronal cell line, Neuroreport 1997; 8: 1481-1484). Currently, maximum levels of transfer expression are reached between 48 and 60 hours after transfection. Therefore, to maximize the ability to detect improved transfection, all assays were performed within 12-20 hours of transfection at a time when reporter gene expression was lower. The authors have previously found that the pDNA: liposome ratio of 1: 3 (w / w) is optimal for transfection in ND7 cells (McQuillin A. et al., Neuroreport 1997; 8; 1.481 - 1484). Therefore, the effect of different amounts of peptide on transfections at 1: 3, 1: 4 and 1: 6 pDNA: CD-Chol / DOPE ratios was compared. Gei retardation analysis showed that the approximate ratio of 1: 0.5 (w / w), pDNA: Mul was sufficient to bind substantially all of the plasmid DNA (Fig. 1). Initial experiments using this ratio and liposomes, however, did not demonstrate transfection efficacy (not shown). The volumes used to form the transfection complexes were much larger than those used to perform the gel retardation test (400 μΐ vs 20 μΐ). Therefore, the authors tested a larger amount of Mul which had a similar concentration in solution as the peptide used in the gel retardation assay. The effect of 0.6, 6, 12 and 21 µg of Mul peptide on DC-Chol / DOPE-mediated transfections was compared. Mul was found to be able to improve cationic liposome-mediated transfection efficiencies by more than 4-fold. The greatest improvement in transfection efficiency was achieved at relative ratios of 1/12/6 pCMVp / Mul / (DC-Chol / DOPE) (w / w / w). This combination resulted in an eleven increase in transfection compared to DNA alone (Fig. 2).

The β-galactosidase reporter gene assay provides a measure of the total level of β-galactosidase generated but does not provide any information regarding the number of cells transfected. For this reason, ND7 cells were also counted in transfected ND7 cells. Cells were seeded at a density of 4 x 10 ⁴ in 24-well culture plates. After 24 hours, the plates were washed briefly with serum-free medium and transfected with pCMVβ complexed with DOCho1 / DOPE and Mul peptide. Optimal ratios determined by the 1: 12: 6 reporter gene assay, pCMVp: Mul: DC-Chol / DOPE were used as ratios. Using these ratios, a 6-fold increase in the number of β-galactosidase positive cells was found (Figures 3 and 6). No apparent cell loss was detected with the Mul complex at any of the concentrations used. Similarly, the protein concentration in the cell lysates used for the β-galactosidase reporter gene assay did not vary compared to untransfected cells (data not shown).

In contrast, no increase in transfection efficiency was observed with Vp1 (Fig. 4). No improvement in transfection efficiency compared to DNA alone was also observed with pCMVβ complexed with Mul alone.

Similar assays on COS-7 cells were performed to determine whether improved transfection in other cell types could be achieved. It also improved liposome-mediated transfection in COS-7 cells (Fig. 5). The same pDNA: Mul: liposome ratio was found to be optimal for both ND7 and COS-7 cells. A similar degree of improvement (3.7-fold) was also observed with respect to cationic liposomes alone.

Example 3

Transfection in differentiated ND7 cells

The inventors also investigated the ability of the Mul peptide to improve cationic liposome-mediated transfection in differentiated ND7 cells. The ND7 cell line is derived from a fusion of rat dorsal root ganglion (DRG) primary neurons and murine neuroblastoma N18Tg2 (Wood JN et al., Novel Cell-Lines Display Properties of Neoconsive Neurons. Proceedings of the Royal Society of London Series B-Biological Sciences 1990; 241: 187-194.

-20EN 298353 B6

ND7 cells can be differentiated in a variety of ways including serum collection, administration of cAMP, or exposure to decreased serum concentration plus cAMP and nerve growth factor. Differentiation of ND7 cells results in the expression of cellular properties associated with other parental nociceptive sensory neurons, including a reduction in cell division and initiation of neurite outgrowth. ND7 cells were seeded into 24-well culture plates and differentiated 24 hours later. 15 to 20 hours after the onset of differentiation, transfection was performed as above. 15-20 hours after transfection, cells were fixed and processed for X-gal histochemical assay. Consistent with previous observations, there was considerable variation in transfection efficiency between the three differentiated groups. ND7 cells differentiated by serum collection showed the lowest transfection rates (1.3%), while the highest rates were observed in the cAMP group (8%) of the low serum / cAMP / NGF group (4.7%) ( Fig. 5). However, under all three conditions, the addition of the Mul polypeptide to the transfection complex improved the transduction of differentiated ND7 cells. ND7 cells differentiated with either cAMP alone or low serum / cAMP / NGF exposure exhibited more than six-fold potency gains (Fig. 5). However, the greatest increase in efficacy was observed in the group of differentiated increases.

Mul and DNA peptide complexes

As shown in Example 1 (DNA binding analysis) using gel electrophoresis, migration of plasmid DNA was significantly reduced and only a small amount of DNA migrated out of the well at a Mul: DNA ratio of more than 0.5: 1.0 (w / w). . This demonstrates that the Mul peptide has strongly interacted with DNA and could neutralize and condense nucleic acids to form small particles suitable for gene delivery. The Mul: DNA particle size (MD) was tested over the Mul: DNA concentration range shown in Figure 7.

The MD particles were prepared by mixing. In fact, appropriate aliquots of the Mul peptide in deionized water were added to plasmid DNA (pCMVβ) (final concentration 220 pg / ml) in 20 mM Hepes buffer, pH 7.0. After mixing well, each mixture was incubated at 20 ° C for 10 min. Immediately after incubation, each mixture was diluted with Hepes buffer (final DNA concentration 24 g / ml) and subjected to particle size analysis by photon correlation spectroscopy (N4 plus, Coulter). All measurements were taken at 20 ° C and data recorded at 90 °. Unimodal analysis was used to calculate mean particle size and standard distribution (S.D.).

Interestingly, although the Mul-bound DNA formed complexes over the entire concentration range tested, the particle size varied significantly depending on the Mul: DNA ratio. Stable, small nanoparticles formed at a Mul: DNA ratio of 0.3 to 1.2 (range L) and greater than 5 (range H). Intermediate ratios resulted in considerable aggregation at which the particle size of the complex increased over the incubation period to reach a size of more than 2 µm (Fig. 7).

Example 4

LMD preparation in the L range

The authors investigated whether liposome-Mul-DNA complexes with a low MD: DNA ratio could form stable nanoparticles and whether the resulting particles would have a good transfection activity complex.

Liposome preparation: DC-Chol (30 pmol) and DOPE (20 pmol) were combined in dichloromethane. The organic solvent was removed under reduced pressure using a rotary evaporator and the residue was dried under vacuum for 3 hours. Then, 4 mM Hepes buffer, pH 7.0 (3 mL) was added to the lipid film with vigorous stirring. After a short sonication (2-3 min), the obtained cationic liposome suspension was extruded three times on an Extruder device (Lipex Biomembranes) through two stacked polycarbonate filters (0.2 µm Millipore) and then ten times over two stacked polycarbonate filters (0 1 µm Millipore) to form small liposomes (mean diameter 109 nm as measured by PCS) (approximately 8 to 10 mg / ml depending on the preparation).

-21 GB 298353 B6

Preparation of liposome: Mul: DNA (LMD) complexes: Mul peptide (0.12 mg in deionized water, peptide concentration 3.5 mg / ml) was added to plasmid DNA solution (pCF1-CAT) (0.2 mg, plasmid concentration) typically 1.0 mg / ml) in 4 mM Hepes buffer with vigorous stirring. A cationic liposome suspension (total lipid amount of 2.4 mg, 4 pmol) was added to the mixture to form small particles with a narrow size distribution (168 nm ± 58 nm) as measured by PCS. These LMDs (final DNA concentration 0.14 mg / ml) were stored at -80 ° C with the addition of 10% sucrose (w / v) until use. No particle size change was observed within one month.

Liposome: DNA (LD) complexes (lipoplexes) were prepared for control experiments using a 3: 1 liposome: DNA (w / w) ratio, which is the optimal composition for transfection of ND7 cells.

ND7 cell transfection: ND7 cells were seeded in normal growth medium (NGM) (with 10% serum) to a density of approximately 4 x 10 ⁴ cells per well, in a 24-well culture plate. After 24 hours, cells were washed by brief exposure to NGM (serum free) medium and then mixed with solutions containing LMD or LD complex previously diluted with NGM (serum free) (in all cases, the final NDA concentration was 3.2 pg / ml) for the indicated time. . The cells were then washed again and incubated before harvesting for an additional 48 hours. The transfection rates were determined by the chloramphenicol transferase (CAT) enzyme assay using ¹⁴ C-CAM as a substrate (Promega). Transfection activity was expressed as a percentage of the conversion of the ¹⁴ C-CAM inserted by the enzyme.

We have found a much higher expression of the reporter gene in LMD-mediated transfection compared to LD. Indeed, LMD transfection resulted in a 6-fold enzymatic activity of CAT after a transfection time of 10 min and a 6-fold activity after a transfection time of 60 min compared to transfection mediated by LD (Fig. 8). Significant transfection was observed for LMD even when the transfection time was only 10 min. These data show how quickly LMD particles are able to enter cells.

Example 5

Changes of cationic lipid (cytofectin)

The inventors investigated whether LMD complexes could be improved by the introduction of polycationic cholesterol lipids (WO 97/45442). CDAN (Β198), ACHx (CJE52) and CTAP (B232) lipids (Fig. 9) were used instead of CD-Chol for the preparation of cationic liposomes. Each cationic liposome system was composed of 60 mole percent cationic lipid and 40 mole percent DOPE and prepared as described in Example 4. The following different LMD complex systems were prepared and compared to each other: LMD (DC-Chol), LMD (B198), LMD (CJE52) and LMD (B232). All LMD systems were prepared with cationic liposomes (total lipid 20 pmol) and 0.6 mg of Mul peptide per 1.0 mg of DNA (pCMVβ) as described above. The particles have been shown to have a diameter of less than 200 nm.

Complex liposome: DNA (LD) (lipoproxy) mixtures were prepared for control experiments with a liposome: DNA ratio of 3: 1 (w / w), which is the optimal composition for transfection of ND7 cells.

ND7 cell transfection: ND7 cells were seeded in NGM medium (with 10% serum) to a density of approximately 4 x 10 ⁴ cells per well, in a 24-well culture plate. After 24 hours, cells were washed by brief exposure to NGM medium (serum free) and then mixed with solutions containing LMD or LD complexes (pre-diluted NGm (serum free)) (final DNA concentration in all cases 2.5 pg / ml) and incubated for 1 hour. The cells were then washed again and incubated

-22GB 298353 B6 48 hours prior to processing for X-gal histochemical staining. The number of blue cells was counted under an inverted microscope.

In all cases, the LMD formulations worked better than the corresponding LD systems prepared with the same polycationic cholesterol lipids (Fig. 10). The order of transfection efficiency was LMD (B198)> LMD (DC-Chol)> LMD (CJE52) LMD (B232). The same order, Β198> DC-Chol> B232, was observed with the corresponding LD systems.

Example 6

The amount of Mul peptide in the range of L

The authors tested the effect of the Mul: DNA ratio (in the L range in Fig. 7) on transfection activity.

Cationic liposomes composed of cationic lipid B198 and DOPE (3: 2 mol / mol) were prepared in the same manner as described in Example 4. A number of complex MD mixtures (Mul: DNA ratio from 0.3 to 1.2) were prepared and were formed by cationic liposome complexes. The obtained LMD systems were composed of liposomes: Mul: DNA (pCMVp) in ratios of 12: 0.3: 1.12: 0.6: 0.6, 12: 0.9: and 12: 1.2: 1 wt. /hmotn./hmotn. The LMD particle sizes measured were approximately 150 nm.

Transfection activities were evaluated in vitro using Panc-1 cells (human pancreatic cancer cell line). Cells were seeded at approximately 5 x 10 ⁴ per well in a 24-well culture plate in RPMI medium supplemented with 10% FCS and grown for 24 hours in the presence of 5% CO ₂ at 37 ° C. Cells were washed by brief exposure to RPMI medium and then mixed with LMD complex solutions pre-diluted with RPMI (final DNA concentration 5.0 µg / ml in all cases) for 30 min. The cells were then washed again and incubated for an additional 48 hours in RPMI medium supplemented with 10% FCS prior to harvesting and assaying for β-galactosidase enzymatic activity using a standard assay kit (Promega).

As shown in Figure 11, the optimal liposome: Mul: DNA ratio for transfection of Panc-1 cells was found to be 12: 0.6: 0.6. Otherwise, LMD complexes with these low levels of Mul achieved excellent transfection results.

Example 7

Quantity and composition of lipids

To determine the effect of changing the ratio of cationic lipid to DOPE as well as the ratio of total lipid to muI and DNA, a number of LMD systems were prepared using B198 as the preferred cationic lipid. Cationic liposomes composed of 60 molar% B198 and 40 molar% DOPE (3: 2 mol / mol), 50 molar% B198 and 50 molar% DOPE (1: 1 mol / mol) and 33 molar% B198 and 67 molar% DOPE were prepared. (1: 2 mol / mol), which were combined with a standard MD complex complex (Mul: DNA 0.6: 1 w / w). in the proportions shown in FIG. 12 as described in Example 4.

Complex liposome mixtures were prepared for control experiments; DNA (LD) (lipoplexes) with a liposome: DNA ratio of 3: 1 (w / w). All LMD systems were found to have a larger average size when lower amounts of cationic liposomes were added to the MD complexes. However, the particle size of the LMDs composed of more than 12 pmol lipids / mg DNA remained less than 200 nm, while the particles containing 12 to 6 pmol lipids / mg DNA were larger in size. In some cases, visible aggregation was observed during the preparation of LMD systems composed of 6 pmol lipids / mg DNA.

-23EN 298353 B6

Transfection activities were determined using Panc-1 (Fig. 12). Cells were seeded at an approximate density of 5 x 10 ⁴ per well in a 24-well culture plate in DMEM medium supplemented with 10% FCS and cultured for 24 hours in the presence of 5% CO 2 at 37 ° C. Cells were washed by brief exposure to DMEM and then mixed with solutions containing LMD or 5 LD complexes diluted with DMEM (final DNA concentration in all cases 5.0 µg / ml for 2 hr). The cells were then washed again and incubated for an additional 48 hours in DMEM supplemented with 10% FDS prior to harvesting and assaying for β-galactosidase enzymatic activity using a standard assay kit (Promega).

The maximum transfection activity was not significantly different in the three formulations tested with different lipid to DOPE ratio. For LMD systems prepared with a B198: DOPE ratio (1: 2 mol / mol), maximum transfection was achieved at the liposome ratio; DNA of approximately 12.5 pmol lipid / mg DNA. The transfection activities of LMD systems prepared with a ratio of B198: DOPE (3: 2 mol / mol) and B198: DOPE (2: 2 mol / mol) reached steady state levels at liposome: DNA ratios above 123.5 pmol lipid / mg DNA. All LMD systems analyzed tended to exhibit low transfection activities at low liposome ratios; DNA (FIG. 12). It is contemplated that LMD formulations composed of low amounts of Mul peptide should need more cationic liposomes compared to formulations prepared using higher amounts of Mul peptide to achieve complete 20 transfection activity for possible LMD systems.

Example 8

Comparison of the Mul peptide with the protamine

Protamine is the occurring cationic peptide that is found in large quantities in 25 fish sperm and has a strong ability to neutralize and condense DNA. Protamine transfection activity was compared to that of the Mul peptide. Peptide Mul or protamine sulfate (Sigma, salmon class X) was complexed with DNA (pCMVp) and then cationic liposomes (B198: DOPE at 3: 2 mol / mol) to give a liposome: peptide: DNA ratio of 12: 0, 6: 1 (w / w / w).

Transfection activities were detected in Swiss 3T3 cells. Cells were seeded at approximately 2 x 10 ⁴ per well in a 24-well culture plate in DMEM supplemented with 10% FCS and cultured for 48 hours to achieve confluency in the presence of 5% CO ₂ at 37 ° C. Cells were washed by brief exposure to DMEM medium and then mixed with solutions containing 35 LMD or LD complexes with pre-diluted DMEM medium (final DNA concentration in all cases).

The cells were then washed again and incubated for an additional 48 hours in DMEM supplemented with 10% FCS prior to harvesting. The degree of β-galactosidase enzymatic activity was determined using a standard assay kit (Promega).

As shown in Figure 13, the Mu 1 peptide complexes showed better transfection of these confluent cells than the protemine complexes.

Example 9

Alternative cationic peptides

A number of liposome: cationic peptide: DNA complexes were prepared to assess the effect of various alternative cationic peptides on transfection activities and their relative transfection capabilities were analyzed in vitro. The peptides were polylysine hydrochloride (average molecular weight 3970, Sigma), polyarginine hydrochloride (average molecular weight 11,800, Sigma), protein derived from protein V, pV (p5, sequence shown below) and peptide analogue Mul (V, sequence shown below) and itself peptide Mul. The peptide p5 peptide and peptide V were synthesized using the same solid phase peptide synthesis method used to prepare the Mul peptide.

-24GB 298353 B6

Each peptide was combined with a cationic liposome (DC-Chol: DOPE 3: 2 mol / mol) and DNA (pCMVp) in a liposome: peptide: DNA ratio of 12: 0.6: 1 (w / w / w) as is as described in Example 4. Transfection activities were tested using HeLa cells (human epithelial cells). These cells were seeded at an approximate density of 5 x 10 ⁴ per well in a 24-well culture plate in DMEM medium supplemented with 10% FCS and cultured for 24 hours in the presence of 5% CO 2 at 37 ° C. Cells were washed by brief exposure to DMEM and then mixed with solutions containing LMD or LD complexes, diluted with OPTIMEM (Gibco) (final DNA concentration in all cases 1.0 pg / ml) for 30 min. The cells were then washed 10 again and incubated for an additional 48 hours in DMEM medium supplemented with 10% FCS before harvest.

The degree of β-galactosidase enzymatic activity was determined by a standard test kit (chemiluminescence, Roche).

As shown in Figure 14, cationic peptides derived from adenovirus (Mul and p5) and analogue Mul 15 (V) showed excellent transfection activity as compared to complexes prepared using synthetic cationic polypeptides, polylysine and polyarginine.

The amino acid sequence of peptides p5, V and Mul.

P5; RPRRRATTRRRTTTGTRRRRRRR

IN; VRRVHHRRRRVSHRRVRGG

Mu 1; MRRAHHRRRRASHRRMRGG

Example 10

Comparison with Transfast using Panc-1

LMD and LD were prepared in the same manner as described in Example 4 except for the use of pCMVβ.

DNA Transfast (Promega) was prepared according to the manufacturer's protocol,

Transfection activities were evaluated in vitro using Panc-1 cells. Cells were seeded at approximately 5 x 10 ⁴ per well in a 24-well culture plate in RPMI Medium supplemented with 10% FCS and grown for 24 hours in the presence of 5% CO ₂ at 37 ° C. Cells were washed by brief exposure to RPMI medium and then mixed with solutions containing LMD or LD complexes diluted with RPMI medium (final DNA concentration in all cases 5.0 µg / ml) for the indicated time. The cells were then washed again and incubated for an additional 48 hours in RPMI 35 supplemented with 10% FCS before harvest. The level of β-galactosidase enzymatic activity was determined using a standard assay kit (Promega). Transfection with Transfast: DNA was performed in serum-free medium (optimal conditions) for 1 hour.

As shown in Figure 15, LMD showed better transfection activity than the Transfast: DNA and 40 LD complex. These results are fully consistent with those found in ND-7 cells.

Example 11

Comparison with Lipofectamine using human bronchial cells

The transfection activity of LMD complexes was compared to that of Lipofectamine (Gibco) complexed with DNA using HBE (human bronchial epithelial cells) cells.

Cells were seeded in a 12-well culture plate in DMEM supplemented with 10% FCS and cultured for 24 hours in the presence of 5% CO ₂ at 37 ° C. Cells were washed by brief 50 exposure to DMEM and then mixed with solutions containing either LMD (preparation as in Example 4) or LD (preparation from lipofectamine: DNA 12: 1 w / w complex), diluted with OPTIMEM medium (Gibco) (final). DNA concentration in all cases

-25GB 298353 B6

5.0 µg / ml) over the indicated times (see Fig. 16). The cells were then washed again and incubated for an additional 48 hours in DMEM medium supplemented with 10% FCS before processing for X-gal histochemical staining.

LMD showed better transfection ability than lipofectamine (Fig. 16) and also showed faster uptake by HBE cells. Similar results were observed in ND7 and Panc-1 cells.

Example 12

Comparison with LT1 using rat brain; expriment ex vivo

The inventors tested transfection activities in organotypic cultures from rat brain using reporter DNA (pCMVβ) to mimic the model in vivo. Brain sections were maintained on transparent porous membranes and were monitored to maintain their internal connectivity and cell architecture to a large extent.

LMD and LD complexes were prepared as shown in Example 4. LT1 is a polyamine transfection reagent manufactured by PanVera Co. A complex containing a cationic liposome (DC Chol: DOPE, 3: 2 mol / mol), LT-1 and plasmid pCMVp at a ratio of 3: 3.2: 1 (w / w / w) was prepared. Brain sections were exposed to the following solutions containing LMD, LD, or liposome: LT1: DNA for 2 hours (Murray et al., Gene Ther, 1999, 6, 190-197). In all cases, no morphological changes in sections were observed during the experiment. After a 48 hour incubation after transfection, cells were harvested, stained with X-gal and the amount of blue cells counted on the slice (Fig. 17).

At a DNA dose of 5.0 µg (2 ml culture), LMD yielded a significantly higher number of blue stained cells than the LD or LT1 complex after X-gal staining. Even at a dose of 129 ng, the LMD complex showed significant transfection activity, even higher than that of the LD complex (DC Chol: DOPE complexed with DNA, 3: 1 w / w ratio) (DNA dose 5.0 µg). The authors found much higher expression of the reporter gene in the LMD complex compared to transfection mediated by the LD and liposome: LT1: DNA complexes. In fact, LMD-mediated transfection was more than nineteen more potent than LT-mediated transfection and more than four times more potent than transfection using liposome: LT1: DNA complex at comparable doses.

Example 13

Comparison with cationic liposomes GL-67; in vivo experiment in mouse lungs

In vivo transfection activity in mouse lung complex LMD (prepared as described in Example 4 using cationic liposomes DC-Chol: DOPE [3: 2, mol / mol] and plasmid pCF1-CAT) was tested and compared to the cationic transfection activity GL-67: DOPE and DMPE-PEG ₅₀ oo liposomes (1: 2: 0.05 mol / mol / mol) complexed with pCF1CAT (LD) liposome: DNA ratio 5.4: 1 w / w) used to enhance the effect in clinical lung trials (Alton et al., Lancet, 1999, 353, 947-954).

LMD (0.14 mg / ml final DNA concentration; 100 ml volume; 14 µg DNA dose) was inhaled into the lungs of Balb / c mice, GL-67: DOPE: DMPE-PEG5000 (1: 2: 0.05 mol / mol) / mol) was complexed with plasmid pCF1-CAT (final DNA concentration 0.8 mg / ml; volume 100 µl; DNA dose 80 µg) and this LD complex was similarly inhaled into the Balb / c lung. After 48 hours, the lungs were homogenized and tested for CAT activity.

The results show (FIG. 18) that LMD and LD system containing GL-67 provided substantially equivalent in vivo transfection rates, although the LMD system delivered a 5-fold lower DNA dose.

-26EN 298353 B6

Example 14

LMD systems with modified sugar

For in vivo applications, non-specific LMD interactions with the biological environment should be minimized. For example, when administered intravenously, obstacles to unwanted interaction with blood components (salts, proteins ...) and non-target cells are important. This opsonization of foreign particles with plasma proteins is one of the first steps in the natural process of removing foreign particles inherited by the immune system. To reduce protein binding and aggregation induced by>

salts can be bound to naturally occurring polysaccharides to LMD. This modification ’

LMD carbohydrate can also be used to direct LMD to carbohydrate receptors.

The neoglycolipids described in Figure 19 have been designed to achieve the desired effect. These compounds are based on three different domains.

ACHx (CJE 52): This lipid (see Figure 9) was chosen as a general lipid platform for the desired neoglycolipids. The cholesterol aliphatic ring system is a very hydrophobic region that is inserted into the lipid envelope of LMD or LD particles and acts as a neoglycolipid anchor.

Carbohydrate motif: The choice of oligosaccharides was limited by the complexity of additional chemical reactions related to carbohydrate modification. The authors decided to use commercially available long-chain carbohydrates, maltotetraose and maltoheptaose, to prove this principle.

Linker: The use of a linker with chemical selectivity has proven effective and flexible and has enabled the synthesis of a wide range of neoglycolipid. This chemoselective technology was based on the conversion of CJE52 to hydroxylaminolipid, which was able to bind directly to unprotected carbohydrates. The synthesis of a typical hydroxylamino-CEJ52 compound is shown in Figure 20, Scheme 1, and the carbohydrate moiety to this linker is based on the glycosylation of O-substituted hydroxylamine (the principle of this reaction with glucose is illustrated in Figure 21, Scheme 2). . Using this strategy, maltotetraose and maltoheptaose were coupled to obtain GLU4 and GLU7 compounds (structure in Figure 22).

This LMD glycomodification was based on the natural ability to dissociate neoglycolipid micelles and incorporate free lipids into LMD membranes. First, LMD complexes were formed from DC-Chol: DOPE cationic liposomes, M11 peptides, and pCMVp plasmid as described in Example 4. Thereafter, a suspension of neoglycolipid micelles in Hepes buffer, pH 7.0, was added to the LMD mixtures and incubated for 30 min. at 20 ° C prior to storage at -80 ° C (Fig. 23).

LMD Neoglycolipid Stabilization: The stabilizing effect of the modified LMD neoglycolipid was evaluated by introducing 7.5 molar% GLU or GLU7 into the LMD. The lipid layer of the LD systems aggregates upon exposure to salt to date. Therefore, a portion of LD (final DNA concentration of 1 pg / ml) was tested for 30 min at 37 ° C in OPTIMEM medium by photon correlation spectroscopy (Photon Correlation Spectroscopy, N4 plus, Coulter). Unimodal analysis was used to evaluate the mean particle size. The average percentage increase in LD particle size is shown in Figure 24. The same procedure was used for the LMD, LMD (GLU4), and LMD (Glu7) base systems (final DNA concentration of 1 μ§ / ιη1),

These results show that LMD is more stable in solution than LD, but show that the presence of GLU4 and GLU7 enhanced the anti-aggregation stabilizing effect on LMD particles at a concentration of 7.5 molar%.

In vitro transfection efficiency: Transfection activity was determined using Hela cells seeded at 5 x 10 ⁴ cells / well in 24-well culture plates and grown to approximately 70% confluency in DMEM supplemented with FCS at 37 ° C and in the presence of

-27GB 298353 B6

5% CO ₂ . Cells were washed in PBS and then exposed to solutions containing LMD complexes pre-diluted with DMEM containing FCS containing the percentages (final DNA concentration in all cases 5.0 pg / ml) for 30 min. The cells were then washed and incubated for an additional 48 hours in normal medium (NGM) before harvesting. The level of β-galactosidase expression was determined by a standard test kit (chemiluminescence, Roche).

The results show an increase in transfection efficiency due to sugar modification at both 0% and 50% serum concentrations (Fig. 25).

Discussion

The authors have previously shown that DC-Chol / DOPE liposomes are effective in transfecting a neuronal derived ND7 cell line (McQuillin A. et al., Neuroreport 8: 1481-1484). DC-Chol has been successfully used outside the CNS in a number of tissues, and clinical trials have been conducted to treat cystic phthbrosis by gene therapy (Caplen NJ et al., Lipsome-Mediated Cftr Gene-transfer to Nasal Epithelium of Patients With Cystic-Fibrosis. 1995; 1:39-46; Nabel G., Chang A., Nabel E., Clinical Protocol: Immunotherapy of malignancy by in vivo gene transfer to tumors (Hum. Gene Ther. 1992 3: 399-410). It has also been shown that DC-Chol liposomes have no cytotoxic side effects (Nabel GJ et al., Direct gene transfer with DNA liposome complexes in melanoma expression, biological activity, and lack of toxicity in humans. Why. Natl. Acad. Sci. USA) , 1993; 90: 11307-11311; Stewart MJ et al., Gene-transfer in vivo with DNA liposome complexes - safety and acute toxicity in mice (Hum. Gene Ther. 1992; 3: 267-275). For these reasons, the authors aim to develop improved formulations of these liposomes for use in nerve cells.

The inventors describe the use of a virus-encoded protein for transfecting cells. It was found that Mul when used in combination with the cationic liposome DC-Chol / DOPE was able to significantly improve cell transfection. This effect was most likely due to Mul's ability to condense pDNA and could be optimized by changing the ratios of polypeptide, pDNA, and cationic liposomes. The increase in transfection efficiency was significantly more pronounced on differentiated cells. As mentioned above, ND7 cells were derived from primary DRG cells. ND7 cell differentiation induces a similar phenotype to their parent peripheral sensory neurons, including induction of neurite outgrowth, decreased overall proliferation, and decreased transfection ability (Wood JN et al., Novel Cell-Lines Display Properties of Nociceptive Sensory Neurons. Proceedings of the Royal Society of London B- Biological Sciences 1990; 241: 187-194; Murray KD et al. DC-Chol / DOPE mediated transfections in differentiated sensory neurons. Increasing transfection efficiency in differentiated ND7 cells may reflect an increase in the ability to promote transfection in primary neurons or in vivo.

The success of non-viral gene delivery means as a useful alternative to viral vector-based systems depends on developing complexes with higher and longer term transfection efficiencies. Because the initial identification of cationic liposomes as a means of transferring genetic material into cells has succeeded, there has been great pressure to develop better formulations of cationic liposomes (Lee RJ Huang L., Lipidic vector systems for gene transfer. Critical Reviews in Therapeutic Drug Carrier Systems 1997; 14: 173 - 206; Felgner PL et al., Lipofection- and Highly Efficient, Lipid-Mediated DNA-Transfection Procedure, Proceedings of the National Academy of Sciences of the United States of America 1987; 7414-7417). Most attempts to improve the cationic liposomes have been based on structural modifications of the molecule itself (Cooper RG et al, Polyamine analogues of 3 beta- [N ^'- (N', N'-dimethylaminoethane) karbamoyljcholesterol (DC-Chol) as agents for gene delivery. Chemistry and European Journal 1998; 4: 137-151). New formulations have been developed with improved efficiency of transfection (Cooper RG et al, Polyamine analogues of 3 beta- [N- (N ^', N'-dirnethylaminoethan) carbomoyl] cholesterol (DC-Chol) as agents for gene deliveiy. Chemistry - A European Journal 1998; 4: 137-151). However, particular cell types behave due to cationic liposome transfection

-28GB 298353 B6 differently. For example, the Mul polypeptide has been found to be superior in enhancing cationic liposome-mediated transfection than Vp1. This is probably due to a higher Mul charge ratio. While both peptides have approximately the same molecular weight, the overall charge ratio of Mul is more than twice that of Vp1 (Table 1). Accordingly, the Mul polypeptide was able to slow the electrophoretic mobility of plasmid DNA to less than 1/60 of the concentration, indicating how firmly Mul is capable of binding DNA. Although a small shift in pDNA motility was detected using 0.25 gg Mul and 1 µg pCMVp complex, after the addition of 0.5 µg Mul, almost all plasmid was captured near the well deposition (Fig. 1). Ratio 0.5 / 1.0 (w / w). Mul to pCMVp corresponds to a molar ratio of 1000/1. Each molecular Mul contains 12 residues that could potentially bear a positive charge. Thus, the theoretical ratio of Mul charge to pCMVp should be 1.6 (12,000 Mul cations per 7500 pMCVP antions). This ratio should completely neutralize the negative charges on pCMVβ and thus completely stop its migration as observed.

A direct comparison between the amount of Mul, which significantly slowed the migration of plasmid DNA, and which therefore optimally increased transfections, could not be made due to a different preparation method. The peptide-pDNA-liposome transfection complexes were prepared in large volumes (see Materials and Methods). Although a 24-fold amount of Mul (12 pg / l pg pCMVp) was required to achieve optimal enhancement of transfection efficiency than was necessary to stop migration on an agarose gel, the concentration in solution was similar (25 mg / ml, pDNA stop; 30 ng / ml, optimal transfection). The presence of Mul also altered the interaction of pDNA with cationic liposomes. The optimal ratio of DC-Chol / DOPE to PCMVP in the presence of Mul was 6/1, which is twice that previously found optimal in nerve cells (McQuillin A. et al., Neuroreport 1997; 8: 1481-1484; Murray KD et al., Cationic liposomemediated transfection in organotypic explant cultures (Gene ther. 1994; 6: 190-197). Theoretically, the amount of Mul used was to completely neutralize the positive charges on pCMVβ, which would prevent the formation of further complexes with DC-Chol / DOPE. Obviously this was not the case because much higher transfection efficiency could be obtained. It is likely that not all possible charged amino acids were protonated under the buffer conditions used. Why more cationic liposomes are needed to improve transfection is not clear and the authors are currently working to clarify this issue.

Finally, mention should be made of the nuclear localization signal contained in the Vpl, Recent Evidence in the Laboratory of the Invention (Unpublished Observations) and Other Authors (LabatMoleur F. et al., Gene Ther., Gene Ther. 1996; 3: 1010-1017; Zabrner J. et al., Cellular and Molecular Barriers to Gene Transfer by a Cationic Lipid, J. Biol. Chem. 1995; 270: 18997-1987; Thierry AR et al., Characterization of liposome- mediated gene delivery: expression, stability and pharmacokinetics of plasmid DNA, Gene Ther., 1997; 4: 226-237; Coonrod A., Li FQ, Horwitz M., On the mechanism of DNA transfection: efficient gene transfer withot viruses. 1997; 4: 1313 - 1321) show that nuclear transport of transfected material may be ineffective in lipofection. For this reason, attempts have been made to precondensate DNA with polycations containing the peptide sequence. are known to have the ability to localize in the nucleus to improve uptake of transfected DNA into the nucleus (Sorgi F.L., Bhattacharya S., Huang L., Protamine sulfate enhancers lipid-mediated gene transfer. Gene Ther. 1997; 4: 961-968; Namiki Y., Takahashi T., Ohno T., Gene transduction of disseminated intraperitoneal tumor using cationic liposomes containing non-histone chromation proteins: cationic liposomal gene therapy of carcinomatosis. Gene Ther. 1998; 5: 240-246; Fritz J. D, et al., Gene transfer into mammalian cells using histone-condensed plasmid DNA. Hum. Gene Ther. 1996; 7: 1395-1404). However, the inventors have found that the more efficient condensation properties of Mul on DNA far outweighed the nuclear localization ability of Vp1 in terms of increased transfection efficiency. Similarly, Fritz et al., Gene transfer into mammalian cells using histonecondensed plasmid DNA. Hum. Gene Ther. 1996; 7: 1395 - 1404, they found no difference

-29EN 298353 B6 in transfection efficiency between recombinant human histone (H1) and a modified version containing the SV40 large T-antigen nuclear localization sequence. Further studies have shown that the presence of NLS improves the accumulation of transfected pDNA in the nucleus, though through specific intracellular biochemical pathways (Sebestyen MG et al., DNA vectory chemistry: the covalent attachments of signal peptide to plasmid DNA. Nta. Biotech. 1998; 16: 80 Hagstrom JE et al., Nuclear Import of DNA in Digitonin-Permeabilized Cells, J Cell Sel. 1997; 110 (Pt 18): 2323-2331).

All publications mentioned herein are hereby incorporated by reference. Various modifications and variations of the disclosed methods and system of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in connection with particular preferred embodiments, it should be understood that the invention as defined in the claims should not be unduly limited to these specific embodiments. Of course, various modifications of the described embodiments of the invention are apparent to those skilled in the art of molecular biology or related fields within the scope of the following claims.

LIST OF SEQUENCES <110> Mitsubishi-Tokyo Pharmaceuticals lne

Tagawa, Toshiaki

Miller, Andrew

Parouzel, Eric

Murray, Karl

Mathews, David

Russell, Willie

Manvell Michelle

Alton, Eric <120> Nucleid Acid Delivery <130> p6592.wo <140> PCT / GB00 / 04767 <141> 2000-12-12 <150> GB 9930533.6 <15l> 1999-12-23 <160> 5 <170 > PatentIn version 3.0 <210> 1 <211> 19 <212> PRT <213> Artificial <220>

<223> Description of Articial Sequence: Polypeptide <400> 1

Met Arg Arg His His Arg Arg Arg Arg Ser Ser Arg Arg Met 5 10 15

Arg. Gly Gly

-30GB 298353 B6 <210> 2 <211> 23 <212> PRT <213> Artificial <220>

<223> Description of Artificial Sequence: Polypeptide <400> 2

Arg Arg Arg Arg · Arg Ala Thr Thr Arg Arg Arg Gly Thr 1 5 10 .15 Arg Arg Arg Arg Arg Arg Arg Arg 20 May <210> 3 <211> 13 <212> PRT <213> Artificial <220> <223> Description of Artificial Sequence: Polypeptide <400> 3 Lys For Arg Lys Leu Lys Arg Val Lys Lys Lys Lys Ly.s 1 5 10 <21.0> 4 <211> 19 Dec <212> PRT <213> Artificial <220> <223> Description of Artificial Sequence: Polypeptide <400> 4 Met Ala Lys Arg Lys Ser Gly Val Ser Lys Cys Glu Thr Lys Cys 1 5. 10 Thr Pro Pro <210> 5 <211> 19 Dec <212> PRT <213> Artifícial <220> <223> Description of Artificial Sequence: Polypeptide <400> 5 Wall Arg Arg Val His His Árg Árg Arg Arg Wall Ser His Arg Arg wall 1 5 10 15 Dec

Arg Gly Gly

Claims (15)

A non-viral nucleic acid delivery vector comprising a fused polypeptide / nucleic acid complex and a cationic lipid, the complex comprising: (a) a selected nucleic acid sequence; and (b) one or more viral combustion nucleic acid polypeptides or other derivatives, wherein the polypeptides or derivatives thereof are capable of (i) binding to a selected nucleic acid sequence; and (ii) condense the selected nucleic acid sequence; and wherein the selected nucleic acid sequence is heterologous to the polypeptide. The vector of claim 1, wherein the at least one polypeptide is an adenoviral nucleic acid packaging polypeptide or derivative thereof. The vector of claim 2, wherein the adenoviral polypeptide is Mul, pV or pVII, or a derivative thereof. The vector of any one of claims 1 to 3, further comprising a polypeptide comprising a nuclear localization sequence. The vector of claim 4, wherein the polypeptide comprising the nuclear localization sequence is adenoviral pV or a derivative thereof. A fused polypeptide / nucleic acid complex comprising a cationic lipid, a polypeptide component, and a nucleic acid component, for use in delivering a nucleic acid component to the nucleus of a eukaryotic cell, wherein (i) the polypeptide component is a viral nucleic acid packaging polypeptide or derivative thereof; (ii) the polypeptide component or derivative thereof is capable of binding to a selected nucleic acid sequence; and (iii) the polypeptide component or derivative thereof is capable of condensing a selected nucleic acid sequence; and wherein the nucleic acid is heterologous to the polypeptide. The complex of claim 6, wherein the at least one polypeptide is an adenoviral nucleic acid packaging polypeptide or derivative thereof. The complex of claim 7, wherein the adenoviral polyieptide is Mul, pV or pVII, or a derivative thereof. The complex of any one of claims 6 to 8, further comprising a polypeptide comprising a nuclear localization sequence. The complex of claim 9, wherein the polypeptide comprising the nuclear localization sequence is adenoviral pV or a derivative thereof. The complex of any one of claims 6 to 10, wherein the ratio of liposome: selected nucleic acid sequence: polypeptide is 2 to 20: 1: 0.5 to 1, preferably 10 to 14: 1: 0.5 to 0.7, more preferably about 12: 1: 0.6. -32GB 298353 B6 12. A method for producing a non-viral nucleic acid delivery vector comprising a cationic lipid and a fused polypeptide / nucleic acid complex comprising the steps of: (a) contacting the selected nucleic acid sequence with a viral nucleic acid packaging polypeptide or derivative thereof, wherein the polypeptide component or derivative thereof is capable of (i) binding to the selected nucleic acid sequence; and (ii) condense the selected nucleic acid sequence; and wherein the selected nucleic acid sequence is heterologous to the polypeptide; and (b) contacting the nucleic acid / polypeptide complex so formed with a cationic lipid. A method for introducing a selected nucleic acid sequence into a eukaryotic cell, comprising contacting the cell with a complex according to any one of claims 6 to 11, wherein the complex comprises the selected nucleic acid sequence. The method of claim 13, wherein the cell is a neuronal cell, a cancer cell, or an epithelial cell. Use of a viral nucleic acid packaging polypeptide or derivative thereof for the production of a nucleic acid delivery vector according to claim 1,